The relationship between physicians' self-reported target fasting blood glucose levels and metabolic control in type 2 diabetes. The QuED Study Group--quality of care and outcomes in type 2 diabetes.

OBJECTIVE: To investigate the relationship between beliefs of physicians relative to intensive metabolic control in type 2 diabetes and levels of HbA1c obtained in a sample of their patients. RESEARCH DESIGN AND METHODS: Physicians' beliefs were investigated through a questionnaire sent to a sample of self-selected clinicians participating in a nationwide initiative aimed at assessing the relationship between the quality of care delivered to patients with type 2 diabetes and their outcomes. At the same time, physicians were asked to collect clinical data on a random sample of their patients, stratified by age (<65 vs. > or = 65 years). Mean HbA1c levels in the study population were thus evaluated according to target fasting blood glucose (FBG) used by their physicians. RESULTS: Of 456 physicians, 342 (75%) returned the questionnaire. Among the responders, 200 diabetologists and 99 general practitioners (GPs) recruited 3,297 patients; 2,003 of whom were always followed by the same physician and 1,294 of whom were seen by different physicians in the same structure on different occasions. Only 14% of the respondents used target FBG levels < or = 6.1 mmol/l, whereas 38% pursued values >7.8 mmol/l, with no statistically significant difference between diabetologists and GPs. The analysis of the relationship between FBG targets and metabolic control, restricted to those patients always seen by the same physician, showed a strong linear association, with mean HbA1c values of 7.0 +/- 1.6 for patients in the charge of physicians pursuing FBG levels < or = 6.1 mmol/l and 7.8 +/- 1.8 for those followed by physicians who used target values >7.8 mmol/l. After adjusting for patients' and physicians' characteristics, the risk of having HbA1c values > 7.0% was highly correlated with physicians' beliefs. Patients followed by different physicians in the same unit showed a risk of inadequate metabolic control similar to that of patients followed by physicians adopting a nonaggressive policy. CONCLUSIONS: Doctors adopt extremely heterogeneous target FBG levels in patients with type 2 diabetes, which in turn represent an important independent predictor of metabolic control. To improve patient outcomes, physicians-centered educational activities aimed at increasing the awareness of the potential benefits of a tight metabolic control in patients with type 2 diabetes are urgently needed.

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein.

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.

A morphometric study of liver cell dysplasia.

Thirty-seven cases of cirrhosis with large liver cell dysplasia (LLCD) were evaluated by morphometric analysis and the results compared with those in 11 cases of hepatitis B surface antigen (HBsAg)-positive cirrhosis, 12 cases of cirrhosis with nodules of active regeneration, 15 cases of hepatocellular carcinomas, and 15 cases of inactive cirrhosis. The nuclear-cytoplasmic, nucleolar-cytoplasmic, and nucleolar-nuclear ratios of LLCD were significantly higher than those observed in all other nonneoplastic groups. Whereas the nuclear-cytoplasmic and nucleolar-cytoplasmic ratios of hepatocellular carcinoma cells were significantly higher than those measured in dysplastic cells, the latter had a nucleolar-nuclear ratio similar to that of neoplastic cells. These results show that, in contrast to previously accepted criteria, the nuclear-cytoplasmic ratio of LLCD is increased and that some morphometric features of LLCD are consistent with its supposed premalignant nature. The usefulness of a morphometric analysis in evaluating any group of abnormal-appearing hepatocytes is stressed.

Pentose phosphate pathway alterations in multi-drug resistant leukemic T-cells: 31P NMR and enzymatic studies.

31P NMR studies were carried out on the parental drug-sensitive human T-lymphoblastoid cell line CCRI-CEM (CEM) and its multi-drug-resistant (MDR) CEM-VBL100 variants, to assess the role of the pentose phosphate (PP) in MDR expression. CEM and CEM-VBL100 were incubated in the presence of 2-deoxyglucose, as recently proposed by our group (Clin. Chim. Acta 208: 39, 1992). Accumulation of 2-deoxyglucose 6-phosphate was much lower in the drug-resistant than in sensitive cells, indicating PP shunt activation in the MDR variants. This result was confirmed by enzymatic analyses, which demonstrated that, with respect to the parental line, the MDR variant was characterized by a) unaltered hexokinase activity; b) higher glucose 6-phosphate dehydrogenase activity; c) increased levels of reduced glutathione and marked increase of glutathione peroxidase activity after cell exposure to an oxidizing agent (tert-butylhydroperoxide). These results support the view that cell detoxification mechanisms mediated by the pentose phosphate pathway may contribute to the expression of MDR in tumours.

The over-expression of P-glycoprotein in K-562 and DAUDI cells, is associated with a high susceptibility to NK and LAK cells.

We evaluated the susceptibility to natural killer (NK) or lymphokine activated killer (LAK) cell-mediated cytolysis of two pairs of drug sensitive/resistant tumor cell lines which were extensively characterized at phenotypic and genotypic level. In the DAUDI cell system, the acquired capability of tumor cell variants to grow in the presence of a relatively high concentration of vinblastine (VBL) is associated with a marked increase to NK and LAK susceptibility. In contrast in the K-562 cell system, no correlation between drug-resistance, P-glycoprotein expression and susceptibility to NK or LAK activity seems to occur.

High frequency production of hybridomas secreting antibodies to cell antigens.

The efficiency of various immunization protocols for the production of hybridomas secreting immunoglobulins specific to cell antigens was evaluated in 15 different fusion experiments. Some of these experiments were performed with splenic B-lymphocytes from mice at different stages of immunization. This approach allowed a dynamic analysis of immunocompetent splenic B-lymphocyte production during the immunization cycle.

Topology of MDR1-P-glycoprotein as indicated by epitope mapping of monoclonal antibodies to human MDR cells.

The MDR1-P-glycoprotein binding sites of three different murine monoclonal antibodies (MM4.17, MM6.15 and MC57), directed towards living, intact human multidrug-resistant cells were investigated in order to study P-glycoprotein topology. By using synthetic peptide scanning, we demonstrated that well-defined regions localized on the predicted first, fourth and sixth extracellular loops are external. On the basis of the structure of MM6.15 epitope, which is distributed on the above three different extracellular loops (and thus is discontinuous), P-glycoprotein molecules result to be differently organized in the lipid bilayer. Moreover, the outcome of the MC57 and MM4.17 epitopes localization experiments, obtained through the use of phage-displayed peptide libraries, represent an additional challenge to the classical 12-transmembrane domain model of P-glycoprotein, since they agree with the novel topography of the molecule (10-transmembrane domain), which was recently proposed on the basis of biochemical and expression studies.

Expression of lymphocyte homing receptor gene is lost in multi-drug-resistant variants of human T-lymphoblastoid CCRF-CEM cells.

The 2.2-kb human cDNA clone PBL32, encoding for the lymphocyte homing receptor (LHR) was used to study the expression of this determinant in multi-drug-resistant (MDR) variants of human T-lymphoblastoid CCRF-CEM (CEM) cells. LHR is significantly associated with the drug-sensitive phenotype, its expression being progressively and quantitatively reduced in MDR variants of CEM cells according to the extent of drug resistance.

P-glycoprotein epitope mapping. I. Identification of a linear human-specific epitope in the fourth loop of the P-glycoprotein extracellular domain by MM4.17 murine monoclonal antibody to human multi-drug-resistant cells.

A new murine monoclonal antibody (MAb), MM4.17, to human multi-drug-resistant (MDR) cells was found to be reactive in an ELISA with a synthetic 16-amino acid peptide selected from the fourth loop of the P-glycoprotein extracellular domain. Immunohistochemistry indicated that this MAb reacted in human tissues in the same pattern as that previously found with other human-specific MAbs to P-glycoprotein. For a precise definition of the MM4.17 epitope, a peptide library consisting of overlapping 4- to 10-mer residues covering the entire P-glycoprotein-fragment was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MM4.17 epitope is constituted by the continuous-linear TRIDDPET amino-acid sequence (residues 750-757 of the human MDRI-P-glycoprotein). The MAb MM4.17 recognizes only the human MDRI-P-glycoprotein isoform, and excess TRIDDPET peptide blocks the binding of the MAb to MDR variants of CEM cells. These results demonstrate that the amino-acid sequence TRIDDPET from the human MDRI gene represents the first continuous-linear epitope identified in the P-glycoprotein extracellular domain.

The gene encoding for MC56 determinant (drug-sensitivity marker) is located on the short arm of human chromosome 11.

A panel of mouse x human B- and T-cell hybrids was analyzed for the expression of MC56 determinant which marks the drug-sensitive state of CEM cells. Karyotypic and phenotypic analyses of the tested clones showed that the expression of MC56 determinant correlated to the presence of human chromosome 11 and segregated concordantly to the epitopes recognized by monoclonal antibodies in the CD44 cluster. By using a particular class of interspecific rodent x human-cell hybrids in which the human genome counterpart is represented in the different clones only by human chromosome 11 or its fragments, we showed that the gene encoding for MC56 determinant is located on the region p13-pter of the short arm of chromosome 11. Therefore, the hypothesized homology between the drug-sensitivity marker MC56 and the CD44 determinant is supported also by gene mapping studies.

P-glycoprotein epitope mapping. II. The murine monoclonal antibody MM6.15 to human multidrug-resistant cells binds with three distinct loops in the MDR1-P-glycoprotein extracellular domain.

A new murine monoclonal antibody (MAb), MM6.15, to human MDR1 P-glycoprotein was found to be reactive in ELISA with synthetic peptides selected from the predicted sequences of the first, fourth and sixth extracellular loop of MDR1-P-glycoprotein. In order to precisely define the MM6.15-binding site, a peptide library of overlapping 5- to 9-mer residues covering the entire sixth extracellular loop of both human and rodent class-1 P-glycoproteins was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MAb MM6.15 reacts only with human synthetic peptides and that the critical component of the MAb recognition is made up of the amino-acid sequence LVAHKL (residues 963-968 of the MDR1-P-glycoprotein) with histidine (H), lysine (K) and possibly leucine (L), key residues of this immunogenic domain.

Identification of a LFA-1 region involved in the HIV-1-induced syncytia formation through phage-display technology.

We have identified a peptide region on CD18 molecule (the beta subunit of the LFA-1 molecule) involved in syncytia formation of HIV-1-infected lymphocytes. Several phage clones mimicking an epitope of the CD18 cell-surface determinant were isolated from two 9-mer random peptide phage-displayed libraries via their binding to the CD18-specific monoclonal antibody (mAb) MHM23, which in in vitro assay inhibits syncytia formation in HIV-1-infected cells. The peptide sequences displayed on phages that blocked immunolabeling of this mAb on LFA-1-expressing cells were used to identify the epitope recognized by mAb MHM23 by sequence comparison. On the basis of this analysis, two peptides which inhibited syncytia formation in HIV-1-infected cells in vitro were synthesized, thus confirming that they mimic a CD18 domain that is involved in this phenomenon. The results here presented highlight the potential of phage-display technology for the study of biological processes at the basis of virus infection, but also suggest new approaches for the therapy of AIDS.

Murine monoclonal antibody recognizing a 90-kDa cell-surface determinant selectively lost by multi-drug-resistant variants of CEM cells.

We describe a murine IgG1 monoclonal antibody (MAb56), specific for a cell-surface protein structure (MC56 determinant) expressed by the human CEM cell line. A large band of approximately 90 kDa was identified as the main specific component of the MC56 determinant. Such a 90-kDa protein is significantly associated with the drug-sensitive phenotype, its expression being progressively reduced quantitatively in multi-drug-resistant (MDR) variants of CEM cells, according to the extent of drug resistance. In addition, the MC56 determinant is expressed de novo in drug-sensitive revertant cell lines derived from MDR cells and unreactive with the MAb56. The MAb56 shows a high affinity towards the immunizing drug-sensitive CEM cell line (Ka = 1.86 x 10(9) L/mole) while not binding to MDR cell variants. The expression of the MC56 molecule on a variety of human cells and tissues makes such a cellular determinant a candidate as a marker for studying the MDR phenomenon both in vivo and in vitro.

Characterization by somatic cell genetics of a monoclonal antibody to the MDR1 gene product (P-glycoprotein): determination of P-glycoprotein expression in multi-drug-resistant KB and CEM cell variants.

We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with multi-drug-resistant (MDR) human cells. Its specificity toward the MDRI gene product (P-glycoprotein) has been demonstrated by the concordant segregation of the MAb57 epitope with the MDRI gene in interspecific mouse x human cell hybrids, and the reactivity of several different MDRI gene-expressing cells with MAb57, particularly insect cells acutely infected with a baculovirus encoding the MDRI gene. MAb57 can be used to detect, by flow cytometry, variations in the relative drug-resistance levels of several MDR KB and CEM cell variants. This immunological probe has also proven useful in selectively destroying MDR target cells in an antibody-dependent cell-mediated (ADCC) assay system as well as in detecting P-glycoprotein expression in normal and malignant tissues and cells.

La ecografía en la terapéutica no invasiva de los quistes hidatidicos intraabdominales / Ultrasonography in the non invasive treatment of intra-abdominal hydatid cysts

La hidatodosis abarca gran parte de la geografía nacional. Constituye un serio problema de salud por el número de casos, morbilidad, alta recurrencia y mortalidad asociadas a la cirugía. La eficacia del albendazol en el tratamiento de los quistes hidatidicos intraabdominales ha sido comprobada, hallándose asociación entre la pérdida de viabilidad y cambios en el aspecto ecográfico. Es así que el concepto terapéutico de la enfermedad ha cambiado, generándose controversias. El propòsito de este trabajo fue evaluar los cambios ecográficos dinámicos y determinar la evolución de los quistes hidatidicos intraabdominales tratados con albendazol. Material y Métodos: Entre 1997 y mayo de 2005 se estudiaron prospectivamente 23 niños con edades comprendidas entre los 2 y 13 años, que cumplimentaron 3 ciclos de albendazol (21 días cada ciclo a 10 mg/Kg/día). Fueron portadores de 43 quistes didatidicos, 40 hepáticos y 3 esplénicos con diámetro longitudinal promedio de 4 cm (r: 2 a 7.4 cm). Criterios de exclusión: quistes hidatidicos, complicados e intolerancia al albenzazol. Las ecografías se realizaron con un equipo Toshiba capasee, provisto de un transductor de 3.5 MHz., al inicio del tratamiento, al mes y luego de cada 3 a 6 meses dependiendo de la respuesta terapéutica. La evolución se definió en términos de involución parcial, total sin respuesta y agravación. Resultados: En un tiempo promedio de 34 meses (r: 4-94m), el 65 por ciento de los quistes involucionaron totalmente, el 25 por ciento remitió parcialmente y el 10% no presento cambios. Los primeros signos de respuesta al tratamiento fueron: desprendimiento de las membranas, reducción del volumen en un 10 por ciento y/o cambios en el contenido. No se observó agravamiento ni complicaciones. Conclusión: el 90 por ciento de los quistes presentó cambios ecográficos, constatándose una involución total en el 65 por ciento.