A Structured Training for Trans Anal Irrigation in Pediatric Patients Improves Outcomes and Reduce Failures: Results of an Interventional Multicenter Prospective Study.

BACKGROUND: Continence issues due to organic causes including previous colorectal surgery or neurological issues might benefit from Transanal irrigation (TAI) that proved to be highly effective but with a number of limitations including a relatively high discontinuation rates. Our study was aimed at evaluating the efficacy of an advanced protocol tailored to each patient to prevent dropout and increase satisfaction, independence, and quality of life. MATERIALS AND METHODS: This was a prospective, interventional, multicenter, nonrandomized study involving children aged 4-18 years with bowel dysfunction unresponsive to conventional treatments who required TAI. TAI was performed in accordance to the best standards of care with a total irrigation volume that was determined based on low emission X-Ray barium enemas performed at the very beginning of the study. All patients underwent training and assessments of continence, patients' perspectives and quality of life were performed at different timepoints from enrollment (T0) up to 6 months since TAI was introduced (T3). RESULTS: A total of 78 patients were enrolled. Male to female ratio was 1.4:1. Mean age at enrollment was 106.1 ± 42.8 months. Discontinuation was reported by 3 patients (3.8 %). Continence, satisfaction and a number of other outcome measures increased from baseline (T0) to the last visit (T3). In particular, mean Rintala total score increased linearly from 7.8 to 14.8 during the study period (T0 to T3 timepoints). On a multivariate analysis, the only parameter that proved to be inversely associated with continence as well as with other outcome measures was the use of laxatives at enrollment and during the study. CONCLUSIONS: This study has demonstrated the high efficacy of this innovative patient-tailored TAI protocol across all assessed scores. Of note, given the negative impact of laxatives, our findings suggest limiting their use in this patient population to further increase the efficacy of the procedure.

Common variants in SOCS7 gene predict obesity, disturbances in lipid metabolism and insulin resistance.

BACKGROUND AND AIMS: Specific Suppressor of Cytokine Signaling (SOCS) members, such as SOCS7, may play a role in the development of insulin resistance (IR) owing to their ability to inhibit insulin signaling pathways. The objective was to explore the association between common variants and related haplotypes in SOCS7 gene and metabolic traits related to obesity, lipid metabolism and IR. METHODS AND RESULTS: 780 unrelated men were included in a cross-sectional study. We selected three tagged SNPs that capture 100% of SNPs with minor allele frequency ≥ 0.10. Analyses were done separately for each SNP and followed up by haplotype analysis. rs8074124C was associated with both obesity (p = 0.005) and abdominal obesity (p = 0.002) and allele C carriers showed, in comparison with TT carriers, lower BMI (p = 0.001) and waist circumference (p = 0.001). rs8074124CC- carriers showed lower fasting insulin (p = 0.017) and HOMA-IR (p = 0.018) than allele T carriers. rs12051836C was associated with hypertriglyceridemia (p = 0.009) and hypertriglyceridemic waist (p = 0.006). rs12051836CC- carriers showed lower fasting insulin (p = 0.043) and HOMA-IR (p = 0.042). Haplotype-based association analysis (rs8074124 and rs12051836 in that order) showed associations with lipid and obesity -related phenotypes, consistent with single locus analysis. Haplotype analysis also revealed association between haplotype CT and both decreased HDL-C (p = 0.026) and HDL-C (p = 0.014) as a continuous variable. CONCLUSIONS: We found, for the first time, significant associations between SOCS7 common variants and related haplotypes and obesity, IR and lipid metabolism disorders.

Investigation of monoclonal antibody dimers in a final formulated drug by separation techniques coupled to native mass spectrometry.

Therapeutic monoclonal antibodies (mAbs) are highly complex proteins that must be exhaustively characterized according to the regulatory authorities' recommendations. MAbs display micro-heterogeneity mainly due to their post-translational modifications, but also to their susceptibility to chemical and physical degradations. Among these degradations, aggregation is quite frequent, initiated by protein denaturation and then dimer formation. Here, we investigated the nature and structure of the high molecular weight species (HMW) present at less than 1% in an unstressed formulated roledumab biopharmaceutical, as a model of high purity mAb. HMW species were first purified through preparative size-exclusion chromatography (SEC) and then analyzed by a combination of chromatographic methods (ion-exchange chromatography (IEX), SEC) coupled to native mass spectrometry (MS), as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and capillary gel electrophoresis under non-reducing conditions. Both covalently and non-covalently bound dimers were identified at a proportion of 50/50. In-depth characterization of the HMW fraction by SEC and IEX hyphenated to native MS revealed the presence of three mAb dimer forms having the same mass, but differing by their charge and size. They were attributed to different compact and elongated dimers. Finally, high-resolution middle-up approaches using different enzymes (IdeS and IgdE) were performed to determine the mAb domains implicated in the dimerization. Our results revealed that the roledumab dimers were associated mainly by a single Fab-to-Fab arm-bound association.

Venous thromboembolism in critically ill patients affected by ARDS related to COVID-19 in Northern-West Italy.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that was first reported in Wuhan, China, and has subsequently spread worldwide. An association between increased venous thromboembolism in patients with pneumonia-related to COVID-19 has not yet been well described. PATIENTS AND METHODS: We aimed to illustrate cases of pulmonary thromboembolism in patients with acute respiratory distress syndrome related to COVID-19 treated in our intensive care unit. The medical records of patients affected by COVID-19 with acute respiratory distress syndrome in our institute from 1/3/2020 to 31/3/2020 were retrospectively reviewed. RESULTS: Our center registered a high prevalence of thromboembolic events among 62 patients affected by acute respiratory distress syndrome related to COVID-19 despite a regular antithrombotic prophylaxis. Out of these, 32 patients were transferred to other hospitals, and 30 were treated in our center. Venous thromboembolism was registered in 12 (19.3%) cases. In particular, 11 diagnoses of pulmonary embolism and 1 diagnosis of deep vein thrombosis were formulated. We described a case series of venous thromboembolism in nine patients treated in our Intensive Care Unit (ICU). Main pulmonary arteries were always involved in these patients. None of them died. CONCLUSIONS: In conclusion, critically ill patients with ARDS related to COVID-19 may have an increased risk of VTE that could be a leading cause of mortality. These patients require a high index of clinical suspicion and an accurate diagnostic approach, in order to immediately start an appropriate anticoagulant treatment.

Quantification of aflatoxin M1 carry-over rate from feed to soft cheese.

From January to December 2016, samples of milk and feeds of dairy cattle were monthly collected. The concentration of mycotoxins in all matrices was determined using the enzymatic immunoassay technique. The average concentration of aflatoxin B1 (AFB1), deoxynivalenol (DON) and zearalenone (ZEA) in feed was 3.01, 218.5 and 467 ug/kg, respectively. The average AFB1 carry-over rate was 0.84% with a variation between 0.05 to 5.93%. Particle size of the feed (P = 0.030) and individual milk production (P = 0.001) affected this rate. Mini-soft cheeses were produced using milk naturally contaminated with aflatoxin M1 (AFM1) as raw material to study its distribution both in whey and in cheese. The average level of AFM1 in milk was 0.014 µg/l. None of milk samples exceeded the maximum level accepted for AFB1 by the Southern Common Market (MERCOSUR) legislation (0.5 µg/l) and only 5.5% of samples exceeded the European Union (UE) regulations (0.05 µg/l). After the cheese elaboration, the concentration of AFM1 was determined in whey and in cheese. The greatest proportion (60%) was detected in whey while 40% AFM1 remained in the cheese. However, the concentration of AFM1 was higher in the cheese compared to the original milk.

Characterization of nanomedicines' surface coverage using molecular probes and capillary electrophoresis.

A faithful characterization of nanomedicine (NM) is needed for a better understanding of their in vivo outcomes. Size and surface charge are studied with well-established methods. However, other relevant parameters for the understanding of NM behavior in vivo remain largely inaccessible. For instance, the reactive surface of nanomedicines, which are often grafted with macromolecules to decrease their recognition by the immune system, is excluded from a systematic characterization. Yet, it is known that a subtle modification of NMs' surface characteristics (grafting density, molecular architecture and conformation of macromolecules) is at the root of major changes in the presence of biological components. In this work, a method that investigates the steric hindrance properties of the NMs' surface coverage based on its capacity to exclude or allow adsorption of well-defined proteins was developed based on capillary electrophoresis. A series of proteins with different molecular weights (MW) were used as molecular probes to screen their adsorption behavior on nanoparticles bearing different molecular architectures at their surface. This novel strategy evaluating to some degree a functionality of NMs can bring additional information about their shell property and might allow for a better perception of their behavior in the presence of biological components. The developed method could discriminate nanoparticles with a high surface coverage excluding high MW proteins from nanoparticles with a low surface coverage that allowed high MW proteins to adsorb on their surface. The method has the potential for further standardization and automation for a routine use. It can be applied in quality control of NMs and to investigate interactions between proteins and NM in different situations.

ß-Hairpin mimics containing a piperidine-pyrrolidine scaffold modulate the ß-amyloid aggregation process preserving the monomer species.

Alzheimer's disease is a neurodegenerative disorder linked to oligomerization and fibrillization of amyloid ß peptides, with Aß1-42 being the most aggregative and neurotoxic one. We report herein the synthesis and conformational analysis of Aß1-42-amyloid related ß-hairpin peptidomimetics, built on a piperidine-pyrrolidine semi rigid ß-turn inducer and bearing two small recognition peptide sequences, designed on oligomeric and fibril structures of Aß1-42. According to these peptide sequences, a stable ß-hairpin or a dynamic equilibrium between two possible architectures was observed. These original constructs are able to greatly delay the kinetics of Aß1-42 aggregation process as demonstrated by thioflavin-T fluorescence, and transmission electron microscopy. Capillary electrophoresis indicates their ability to preserve the monomer species, inhibiting the formation of toxic oligomers. Furthermore, compounds protect against toxic effects of Aß on neuroblastoma cells even at substoichiometric concentrations. This study is the first example of acyclic small ß-hairpin mimics possessing such a highly efficient anti-aggregation activity. The protective effect is more pronounced than that observed with molecules which have undergone clinical trials. The structural elements made in this study provide valuable insights in the understanding of the aggregation process and insights to explore the design of novel acyclic ß-hairpin targeting other types of amyloid-forming proteins.

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography.

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.

Vaccination: Developing and implementing a competency-based-curriculum at the Medical Faculty of LMU Munich.

BACKGROUND: In Germany medical students should gain proficiency and specific skills in the vaccination field. Especially important is the efficient communication of scientific results about vaccinations to the community, in order to give professional counseling with a complete overview about therapeutic options. AIM OF THE PROJECT: The aim of this project is to set up a vaccination-related curriculum in the Medical Faculty at the Ludwig-Maximilians-University in Munich. The structure of the curriculum is based on the National catalogue for competency-based learning objectives in the field of vaccination (Nationaler Kompetenzbasierter Lernzielekatalog Medizin NKLM). Through this curriculum, the students will not only acquire the classical educational skills concerning vaccination in theory and practice, but they will also learn how to become independent in the decision-making process and counseling. Moreover, the students will become aware of consequences of action related to this specific topic. METHODS: According to defined guidelines, an analysis was performed on courses, which are currently offered by the university. A separate analysis of the NKLM was carried out. Both analyses identified the active courses related to the topic of vaccination as well as the NKLM learning objectives. The match between the topics taught in current courses and the NKLM learning objectives identified gaps concerning the teaching of specific content. Courses were modified in order to implement the missing NKLM learning objectives. RESULTS: These analyses identified 24 vaccination-related courses, which are currently taught at the University. Meanwhile, 35 learning objectives on vaccination were identified in the NKLM catalogue. Four of which were identified as not yet part of the teaching program. In summary, this interdisciplinary work enabled the development of a new vaccination-related curriculum, including 35 learning objectives, which are now implemented in regular teaching courses by the Medical Faculty. CONCLUSIONS: This project successfully describes a method to develop and implement a competency-based teaching program on the topic of vaccination. Importantly, the process presented here can serve as a guide to develop and implement similar teaching programs on other subjects and Universities.

In vitro synergism between 5-fluorouracil and natural beta interferon in human colon carcinoma cells.

The combination of 5-fluorouracil (FUra) plus IFN-beta was studied in vitro using a human colon carcinoma cell line, HCT-8. Continuous exposure to high concentrations of IFN-beta is cytotoxic to these cells (ED50, 600 +/- 50 IU/ml). A strong synergism (P < 0.002) was observed when a short-term (1-h), high-concentration exposure to fluoropyrimidine (300 or 1000 microM) was followed by IFN-beta given continuously. In fact, the mean ratio between the expected (product of the survival of each agent alone) and the observed clonogenic cell survival rates of the combination was 3.4 (range, 2.4-4.9). Longer exposures to the fluoropyrimidine (24 h, 7 days) produced less than additive effects with IFN-beta, indicating strong schedule dependency for this synergism. The mechanism of interaction was studied at four levels. First, thymidylate synthase (TS) activity, inhibition, and recovery were measured by an in situ assay in cells treated with FUra, IFN-beta, and their combination. When the prolonged infusion of IFN-beta followed a 1-h exposure to FUra, the observed TS inhibition and recovery, at each time point, were very similar to the expected values, indicating that the interactions between these drugs at the level of TS are not the determinant of the synergism. Second, cell cycle analysis was done. During the continuous exposure to IFN-beta, a significant accumulation of HCT-8 cells in S-phase was observed at 24, 48, and 72 h compared to untreated controls (P = 0.003); however, under the same experimental conditions producing synergy in the clonogenic assay, no significant cell cycle perturbations were produced by the combination of FUra followed by IFN-beta compared to those caused by each agent alone. Third, using the alkaline elution test, we also demonstrated that the synergism is not due to enhanced FUra-induced DNA single-strand break frequency in high molecular weight DNA. Finally, nucleic acid incorporation experiments, using tritiated FUra, showed that the cytokine, given continuously (300 IU/ml), enhanced the amount of FUra incorporated into nucleic acids 24 h after a 1-h exposure to 300 and 1000 microM of FUra. The median percentage of enhancement values were 31.6 +/- 11.5%,m for the lower drug concentration and 18. 4 +/- 8.1% for the higher drug concentration tested. These results suggest that the mechanism of this synergism may be related to the ability of IFN-beta to promote the incorporation of intracellular FUra metabolites into nucleic acids and/or to inhibit the cleavage of FUra nucleotides from RNA/DNA.

Melatonin reduces the need for sedation in ICU patients: a randomized controlled trial.

BACKGROUND: Critically ill patients suffer from physiological sleep deprivation and have reduced blood melatonin levels. This study was designed to determine whether nocturnal melatonin supplementation would reduce the need for sedation in patients with critical illness. METHODS: A single-center, double-blind randomized placebo-controlled trial was carried out from July 2007 to December 2009, in a mixed medical-surgical Intensive Care Unit of a University hospital, without any form of external funding. Of 1158 patients admitted to ICU and treated with conscious enteral sedation, 82 critically-ill with mechanical ventilation >48 hours and Simplified Acute Physiology Score II>32 points were randomized 1:1 to receive, at eight p.m. and midnight, melatonin (3+3mg) or placebo, from the third ICU day until ICU discharge. Primary outcome was total amount of enteral hydroxyzine administered. RESULTS: Melatonin treated patients received lower amount of enteral hydroxyzine. Other neurological indicators (amount of some neuroactive drugs, pain, agitation, anxiety, sleep observed by nurses, need for restraints, need for extra sedation, nurse evaluation of sedation adequacy) seemed improved, with reduced cost for neuroactive drugs. Post-traumatic stress disorder prevalence did not differ between groups, nor did ICU or hospital mortality. Study limitations include the differences between groups before intervention, the small sample size, and the single-center observation. CONCLUSION: Long-term enteral melatonin supplementation may result in a decreased need for sedation, with improved neurological indicators and cost reduction. Further multicenter evaluations are required to confirm these results with different sedation protocols.

A multi-mode chromatographic method for the comparison of the N-glycosylation of a recombinant HIV envelope glycoprotein (gp160s-MN/LAI) purified by two different processes.

The glycosylation pattern of a recombinant gp160s-MN/LAI variant of human immunodeficiency virus type 1 (HIV-1) was studied in relation to two alternative purification techniques one of which involves an immunoprecipitation step. For analysis a multi-mode high performance liquid chromatography (HPLC) method which combines gel permeation chromatography on the RAAM 2000 GlycoSequencer, weak anion exchange chromatography and normal phase chromatography was developed and profiles were obtained for the fluorescently-labelled glycans released from the two gp160s-MN/LAI preparations. Charged glycans accounted for 77 and 80% of the total glycans for the IAP- and SP-purified samples, respectively. The acidic character of these glycans was mainly due to the presence of sialic acids. However, following sialidase treatment, residual charged glycans were still found. No differences were found in the glycan distributions of the two gp160s-MN/LAI preparations either in their degree of sialylation or in their relative proportion of each separated structure. Although this did not reach statistical significance, a lower proportion of large glycan structures regardless of their charge status was found on the gp160s-MN/LAI prepared by the procedure which involved an immunoaffinity chromatography step.

Insufficient adaptation of hypoglycaemic threshold for cognitive impairment in tightly controlled type 1 diabetes.

UNLABELLED: It is well known that hypoglycaemic thresholds for hormones and symptoms occur at lower plasma glucose levels in patients with strict glycaemic control. However, whether the threshold for cognitive impairment also shifts is still an unresolved question. We studied 19 type 1 diabetic patients, including 8 with hypoglycaemia unawareness, aged 37.0 +/- 7.4 y.r., with diabetes duration 15.2 +/- 10.7 yr, and HbA1c 7.6 +/- 1.1%. Hypoglycaemic thresholds for hormones, symptoms, awareness and cognitive function using the 4-choice reaction time test (4RT), were measured every 30 min during a 150 min stepped 4.4 to 2.2 mM hypoglycaemic hyperinsulinemic clamp. We found that 4RT- accuracy deteriorated earlier than 4RT-time (3.2 and 2.7 mM, respectively, p<0.01), and that both correlated poorly with HbA1C before and after adjustment for age and diabetes duration (r=0.11, and 0.18, respectively). On the opposite, adrenaline, autonomic and neuroglycopenic symptoms, and awareness significantly correlated with HbA1c values (r=0.56, 0.70, 0.61, and 0.63, after adjustment, respectively). Furthermore, after allocating the patients into two subgroups according to HbA1c values (<8% n=12, and >=8% n=7), we found that, as opposed to other thresholds, accuracy and 4RT-time were minimally and not significantly influenced by glycaemic control, therefore exhibiting the smaller glucose thresholds shifts (- 0.2 and - 0.5 mM for accuracy and time, respectively, vs. 0.6 -0.8 for other thresholds). IN CONCLUSION: 1) the hypoglycaemic thresholds for cognitive dysfunction shift with strict glycaemic control, but not significantly and less than other thresholds, 2) as opposed to other reports, accuracy deteriorates earlier than speed during the 4RT test, and 3) these "maladapted" reactions may contribute to the higher risk for severe hypoglycaemia in subjects with tight glycaemic control.

Effects of dipeptide administration on hypoglycaemic counterregulation in type 1 diabetes.

OBJECTIVES: To investigate if a dipeptide made of glutamine and alanine is able to contribute to the recovery from insulin-induced hypoglycaemia in type 1 diabetes. RESEARCH DESIGN AND METHODS: Fifteen adult type 1 patients were randomly assigned to study group (n=7): intravenous infusion of 20 g Dipeptiven in normal saline (i.e., 8 g alanine and 13 g glutamine), or control group (n=8): same infusion, normal saline only. A 150 min gradual hypoglycaemic hyperinsulinemic clamp was administered after 2 h of infusion. Counterregularory hormones, symptoms, and cognitive function (4 choice reaction test) were regularly measured during the study. RESULTS: Blood glucose and glucose infusion rates were similar in the 2 groups. Circulating levels of alanine and glutamine peaked at 90 min and remained elevated throughout the test. This was associated with significant differences in: glucagonemia 107 +/- 20 vs 58 +/- 8 pg/ml, and neuroglycopenic symptoms scores: 7 +/- 3 vs 18 +/- 13, at t 150 min, in study and control group, p<0.05. Dysautonomic symptoms, cognitive tests as well as epinephrine, norepinephrine, cortisol and growth hormone were similar between groups. CONCLUSION: Intravenous infusion of a dipeptide made of alanine and glutamine is capable to reactivate glucagon secretion during insulin-induced hypoglycaemia and to reduce hypoglycaemic symptoms.

One-step capillary isoelectric focusing for the separation of the recombinant human immunodeficiency virus envelope glycoprotein glycoforms.

One-step capillary isoelectric focusing was investigated as a rapid method to resolve the glycoforms of the heterogeneous recombinant human immunodeficiency virus (HIV) envelope glycoprotein (rgp 160sMN/LAI). The separation was performed in a poly(vinyl alcohol) (PVA) coated capillary using a mixture of ampholyte of narrow and wide pH range. A combination of saccaharose and 3-(cyclohexylamino)-1-propanesulfonic acid was shown to be the most efficient additive to avoid protein precipitation which occurs at a pH close to its pI. Although the calibration curve [isoelectric point (pI) vs. migration times] showed a non-linear relationship, an adequate linearity could be yielded for short pI ranges permitting to exhibit the acidic character of the different glycoforms of the rgp 160s MN/LAI (pI from 4.00 to 4.95). Reproducibility evaluated by comparing the performance of a polyacrylamide and a PVA coated capillary showed that low RSD values were obtained for intra-day (0.5 to 1.9%) and inter-day (1.6 to 7.6%) measurements using the PVA capillary. Moreover, the long term stability of the PVA capillary was demonstrated by measuring the variation of migration times of the protein markers for a long period of use. Finally, this method was able to differentiate the glycoform pattern of two close glycoproteins such as the rgp 160 of two sub-populations of the virus HIV-1.

Routine o-glycan characterization in nutritional supplements--a comparison of analytical methods for the monitoring of the bovine kappa-casein macropeptide glycosylation.

Analytical procedures, including capillary isoelectric focusing (CIEF), high-performance anion-exchange chromatography coupled to amperometric detection (HPAEC-PAD) and normal-phase chromatography with fluorescence detection are presented for the characterization of a highly O-glycosylated caseinomacropeptide (CGMP) and the detection of subtle glycosylation differences between CGMP Batches obtained with two different preparation procedures. Modified two-step CIEF allowed monitoring of glycopeptide heterogeneity and determination of the isoelectric points of acidic glycoforms. The mixture of wide and narrow pH range ampholytes was optimized to improve glycoform resolution. The pI of the different CGMP glycoforms was evaluated with pI internal standards and found to range between 3.08 and 3.58, which indicates a very acidic glycopeptide. Moreover, the monosaccharide composition was determined with HPAEC-PAD after neutral and amino sugars release by using adequate acidic hydrolysis of CGMP. Results indicated a similar composition for Batches I and II, but the monosaccharide percentages were 3-4 fold higher in Batch I, particularly for galactose and glucose. This likely reflects a higher content in lactose in the case of Batch I. Finally, O-linked oligosaccharides were released with an automated hydrazinolysis and derivatized with a sensitive labelling reagent, 2-aminobenzamide. The derivatives were then analyzed by normal-phase HPLC coupled with fluorescence detection, and separated on the basis of hydrophilic interaction, which allowed oligosaccharide mapping of the two CGMP. It appeared that the two CGMP preparations had an almost identical O-glycan population, but CGMP Batch I was more glycosylated than Batch II. Additionally, the sizes of the separated glycans, expressed as the number of glucose units, were tentatively assigned using calibration with a partial hydrolysate of dextran. In conclusion, a combination of electrophoretic and chromatographic techniques was found powerful in studying glycoprotein heterogeneity and assessing batch-to-batch consistency.

Physicochemical characterization of different batches of ethylated beta-cyclodextrins.

Different ethylated beta-cyclodextrin (Et-beta-CD) derivatives were obtained by various synthetic routes and their chemical structures and physical properties were elucidated. The aqueous solubility studies were carried out at 25 and 37 degrees C. A gas phase chromatography analysis, with head space extraction, was developed to detect the presence of residual solvents in the dry preparations. Electrospray ionization mass spectrometry allowed the determination of the average degree of substitution and the molecular mass of the Et-beta-CDs. Nuclear magnetic resonance analysis was used to elucidate the relationship between the solubility behavior and substituted positions of ethyl groups on the CD glucopyranose units. Finally, this paper deals with different physico-chemical methods used to fully characterize the different batches of Et-beta-CD to correlate the data obtained with the pharmacotechnical behavior of these cyclodextrins.

Contribution of capillary zone electrophoresis to the analysis of cecal mucins.

The ability of capillary electrophoresis to perform the separation of mucin glycoforms has been investigated. Adsorption of mucins to the capillary was observed in most cases, leading to unreproducible results. This was due in part to the characteristic structure of mucin (highly charged, large size) and also to its poor solubility. Various buffers were therefore investigated; it was found that a zwitterionic electrolyte, such as a (3-[cyclohexylamino]-1-propanesulfonic acid) (CAPS) buffer, pH 8.8, greatly improved the separation. Using this buffer, mucin was resolved into five main fractions. The use of several additives, such as cationic molecules (1,4-diaminobutane [DAB]) or hydrophilic polymers (hydroxypropylmethylcellulose [HPMC], polyethylene glycol [PEG]) was also investigated. PEG and HPMC did not affect the separation and the electroosmotic flow (EOF) in the same manner. The favorable effect of the addition of PEG was clearly demonstrated and it was postulated that some interaction of this polymer with the mucins occurred. Finally, the application of the method to the comparison of glycoform patterns of mouse cecal mucins showed a marked difference for mucins derived from two sources: germ-free and gnotobiotic mice. These results indicate that mucins from gnotobiotic mice have been degraded due to the glycosidic activity of the bacterial strains present in these mice.

[Genetics of diabetic complications: retinopathy]. / Génétique des complications du diabète: rétinopathie.

Multiple clinical and physiopathological studies as well as genetic analysis, suggest that diabetic retinopathy (DR) is a consequent of interactions between environmental factors, especially hyperglycaemia, and several genetic factors. The genes of aldose reductase (AR), inducible nitric oxide synthase (NOS2A), endothelial nitric oxide synthase (NOS3), vascular endothelial growth factor (VEGF), pigmented epithelium-derived factor (PEDF), protein kinase C-beta (PKC-beta) and receptor for advanced glycation end products (RAGE) implicated in the pathogenesis of DR. The only genetic marker associated with risk of DR in several studies is a microsatellite (A-C)n at 5'end of AR. The synergistic combination of conventional approaches (e.g. candidate gene association studies) with new emerging technologies (e.g. biochips) will be a key factor in the elucidation of the genetic aspects of DR.

[Acquired partial lipodystrophy. Insulin resistance, hepatic lipase activity and small and dense LDL particles]. / Lipodistrofia parcial adquirida. Resistencia insulínica, actividad lipasa hepática y partículas LDL pequeñas y densas.

Partial lipodystrophy (PLD) is an infrequent condition characterized by symmetric loss of subcutaneous adipose tissue in the upper or lower part of the body, although occasionally it affects only the extremities. In all cases it appears along with acantosis nigricans (AN), insulin resistance and impairment in the metabolism of lipids and carbohydrates. The case depicted pertains to a 49 year old female with no family history involving loss of adipose tissue in face and upper body. No fat in lower part of body was observed. The patient showed facial thinning at age 8, AN at 11 and gestational diabetes during her fourth pregnancy at 33. At present, the patient presents severe hyperglycemia and hyperinsulinemia with a marked insulin resistance. Type IV hyperlipoproteinemia (OMS), declined C-HDL and Apo A1 and low C-LDL but with a high proportion of small and dense LDL particles were present. Non esterified fatty acids were high. Lipoprotein lipase and hepatic lipase activities are in the lower limit and increased respectively. Fraction C3 of the complement was diminished. No mutations were observed either in codons 170, 809 and 972 of the IRS-1 receptor or in codon 276 of the adrenergic beta 2 gene.