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Biomedical subjects

C Nicolau

Publications and source records attributed to C Nicolau.

At least 37 records · Page 2Linked to original sources
PubMed1997

Diagnosis of human immunodeficiency virus infection using citrated whole blood.

Standard isolation of human immunodeficiency virus type 1 (HIV-1) from peripheral blood mononuclear cells (PBMC) requires 5 to 20 ml of blood, and the centrifugal separation of PBMC is expensive and time-consuming. Whole-blood coculture techniques use small sample volumes, do not require centrifugation, and allow measurement of the total viral burden in peripheral circulation. We compared the results of citrated whole-blood coculture with those obtained by the standard AIDS Clinical Trials Group PBMC semiquantitative culture method and reverse transcription-PCR quantitation of plasma HIV-1 RNA levels. PBMC cocultures were also set up with added erythrocytes (RBCs) to determine if the presence of RBCs affects the replication of HIV-1 in vitro. The mean number of cells required for a p24-positive PBMC coculture was approximately seven times greater than that required for a positive citrated whole-blood coculture (P < 0.01). At volumes of 100, 50, and 25 microl, the sensitivities of the whole-blood coculture were 94.5, 93.6, and 87.3%, respectively. The PBMC culture in the presence of added RBCs was more sensitive than PBMC coculture alone. The citrated whole-blood coculture was simple to perform, produced a reliable diagnosis of HIV infection in adult volunteers, was more sensitive than previously reported techniques even in half the culture time, and showed less variability than the PBMC coculture. Citrated whole-blood coculture may be a useful and efficient tool for diagnosing infection with HIV-1.

Adult↗
PubMed1997

Ultrasonographic study of pancreatic cysts in autosomal dominant polycystic kidney disease.

Pancreatic cysts are an uncommon extrarenal clinical feature of autosomal dominant polycystic kidney disease (ADPKD). The prevalence of pancreatic cysts, sonographically assessed in ADPKD and in the different typs of ADPKD (PKD1 and PKD2) has not been reported. We have studied 173 ADPKD patients and 160 non-affected family members and found a prevalence of pancreatic cysts, of 9% in ADPKD patients over 30 years of age. The presence of pancreatic cysts was related to the increasing age, to the female sex and to the type of ADPKD, found exclusively in PKD1 patients. No complications related to pancreatic cysts were recorded. Pancreatic cysts are an unusual feature of ADPKD and do not appear to contribute to morbidity or mortality.

Adolescent↗
PubMed1996

In vivo gene electroinjection and expression in rat liver.

In vivo targeted gene transfer by non-viral vectors is subjected to anatomical constraints depending on the route of administration. Transfection efficiency and gene expression in vivo using non-viral vectors is also relatively low. We report that in vivo electropermeabilization of the liver tissue of rats in the presence of genes encoding luciferase or beta-galactosidase resulted in the strong expression of these genetic markers in rat liver cells. About 30-40% of the rat liver cells electroporated expressed the beta-galactosidase genetic marker 48 h after electroporation. The marker expression was also detected at least 21 days after transfection at about 5% of the level 48 h after electroporation. The results indicate that gene transfer by electroporation in vivo may avoid anatomical constraints and low transfection efficiency.

Animals↗
PubMed1996

Human erythrocytes bearing electroinserted CD4 neutralize infection in vitro by primary isolates of human immunodeficiency virus type 1.

Human erythrocytes bearing electroinserted full-length CD4 (RBC-CD4) can bind and fuse with a laboratory strain of human immunodeficiency virus type 1 (HIV-1) or with T cells infected by HIV-1. Here we show that RBC-CD4 neutralize primary HIV-1 strains in an assay of cocultivation of peripheral blood mononuclear cells (PBMC) from HIV-1-infected persons with uninfected PBMC. RBC-CD4 inhibited viral p24 core antigen accumulation in these cocultures up to 10,000-fold compared with RBC alone. Viral p24 accumulation was inhibited equally well when measured in culture supernatants or in call extracts. The inhibition was dose-dependent and long-lived. Two types of recombinant CD4 tested in parallel were largely ineffective. The neutralization of primary HIV-1 by RBC-CD4 in vitro was demonstrated in PBMC cultures from 21 of a total of 23 patients tested at two independent sites. RBC-CD4 may offer a route to blocking HIV-1 infection in vivo.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Linkage, clinical features, and prognosis of autosomal dominant polycystic kidney disease types 1 and 2.

Linkage analysis was performed on 49 Catalan families with autosomal dominant polycystic kidney disease obtained via the Nephrology Department and related nephrology centers. A total of 336 subjects, 267 at risk for the disease, were investigated using three microsatellites linked to polycystic kidney disease Type 1 (PKD1) and three microsatellites linked to PKD2. All of the subjects underwent physical and sonographic examination. The results demonstrate locus heterogeneity, with 0.85 as the maximum likelihood for the proportion of families linked to PKD1. All of the remaining families were found to be linked to PKD2. Analysis of clinical data in the PKD1 group (N = 146) versus the PKD2 group (N = 20) showed a milder form of the disease in the latter, with a later age at diagnosis (27.4 versus 41.4 yr, P = 0.0002), later age of onset of ESRD (53.4 versus 72.7 yr, P < 0.0001), later age of diagnosis of hypertension (34.8 versus 49.7 yr, P = 0.001) and lower prevalence of hypertension at younger ages. Sonographic findings did not differ significantly between both groups. Although anticipation was observed in both groups, it did not affect the majority of families. No signs of imprinting were found in this study, and the only gender effect was an earlier age of onset of ESRD in men than in women (49.5 versus 53.1 yr in PKD1, P < 0.01 and 70.57 versus 73.6 yr in PKD2, P = 0.1). Molecular analysis of autosomal-dominant polycystic kidney disease allows presymptomatic diagnosis in individuals younger than age 30, and helps in establishing prognosis.

Adult↗
PubMed1996

Abdominal aortic aneurysms and autosomal dominant polycystic kidney disease.

Although cases of autosomal dominant polycystic kidney disease (ADPKD) associated with abdominal aortic aneurysm have been repeatedly reported in the literature, no systematic studies of the aortas of these patients have been performed. In the study presented here, a sonographic study of the abdominal aorta in 139 ADPKD patients and in 149 healthy family members was carried out. For both groups, an increase in aortic diameter related to age and sex, (being wider in men than women) was found. In ADPKD patients, neither a wider aortic diameter nor a higher prevalence of abdominal aortic aneurysms could be found in any age group. It was concluded that, although these patients are prone to develop aortic aneurysms because of hypertension and associated connective tissue disorders, the presence of abdominal aortic aneurysms should be questioned as a frequent feature of ADPKD.

Adolescent↗
PubMed1996

Human insulin-induced lipoatrophy. Successful treatment using a jet-injection device.

OBJECTIVE: To evaluate the efficacy of the administration of insulin by a jet-injector device in stopping and reversing severe human insulin-induced lipoatrophy. CASE: We report a case of a woman with severe human insulin-induced lipoatrophy who has been treated exclusively with recombinant DNA human insulin since the onset of IDDM. RESULTS: The loss of subcutaneous tissue in the injection areas was demonstrated and measured by high-frequency ultrasound. Dermatologic exam demonstrated a severe reduction of fat tissue. After 8 months of administration of human insulin by a jet injector, there were no more new lesions of lipoatrophy and those affected areas were substantially ameliorated. CONCLUSIONS: Jet-injection devices might constitute a helpful method to treat those patients affected by severe human insulin-induced lipoatrophy.

Adult↗
PubMed1995

Modulation of CD4 lateral mobility in intact cells by an intracellularly applied antibody.

This study shows that the lateral mobility of CD4, an important plasma-membrane immune receptor, can be modulated by intracellular application of an anti-CD4 antibody. For this purpose, (i) full-length CD4 and a truncated CD4 mutant, lacking a 32-residue-long C-terminal intracellularly exposed domain, were expressed in Spodoptera frugiperda (Sf9) insect cells, (ii) a monoclonal antibody, C6, with specificity for the C-terminal domain was generated, and (iii) a versatile apparatus for fluorescence microphotolysis (FM) studies was constructed. By these means it was found that the commercial anti-CD4 antibody Leu3a-PE, in contrast with several other anti-CD4 antibodies, could be used as a fluorescent label of CD4 without interfering greatly with CD4 mobility. Labelled by Leu3a-PE, full-length CD4 had a lateral diffusion coefficient of D = (4.7 +/- 1.9) x 10(-10) cm2/s and a mobile fraction of fm = 80 +/- 16% (room temperature). Within experimental accuracy the truncated CD4 had the same mobility as full-length CD4. Introduction of the C6 antibody into Sf9 cells by microinjection or by fusion with C6-loaded liposomes decreased the mobility of full-length CD4 (fm = 40%) but not of truncated CD4 (fm = 80%). Treatment of Sf9 cells with phorbol ester also reduced the mobility of full-length CD4 (fm = 50%) but not truncated CD4 (fm = 90%). A calmodulin inhibitor but not a protein kinase C (PKC) inhibitor abolished the phorbol ester effect.

Amino Acid Sequence↗
PubMed1995

Immune response against the murine mdri protein induced by vaccination with synthetic lipopeptides in liposomes.

Intrinsically, or after exposure to chemotherapeutic drugs, many cancer cells overexpress a class of high molecular weight membrane glycoproteins associated with the multidrug resistance (mdr) of these cells. This report describes an immunization protocol eliciting autoantibodies specific to extracellular epitopes of the murine mdr 1 P-glycoprotein (Pgp). Synthetic peptides with the sequences of extracellular loops of murine Pgp were covalently coupled with four palmitic acid moieties per peptide molecule. These "lipopeptides" were reconstituted in the bilayer of liposomes containing lipid A and used to immunize mice. Antibodies against the lipopeptides corresponding to loop 2 and 4 were elicited in sera of immunized mice. They reacted specifically with extracellular epitopes of the naturally occurring murine Pgp. After interaction with resistant cancer cells, the antibodies induced an average 50% increase in cellular accumulation of doxorubicin and Bodipy-verapamil. In the presence of these antibodies the resistance of L1210 mdr cells was reduced from an LD50 of 4 x 10(-5) M to 5 x 10(-7) M doxorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Life span of circulating membrane CD4 inserted into the plasma membranes of autologous red blood cells of HIV-infected subjects.

Membrane recombinant CD4 was electroinserted into the plasma membrane of red blood cells (RBCs) from four HIV patients. CD4 had been labeled with 125I before electroinsertion. The RBCs-CD4-125I were labeled with 51Cr and autotransfused to the donor patients. The hematological indexes and the P50 value of the RBCs were not modified by the electroinsertion of CD4. The life span of the RBCs was not affected by electroinsertion of CD4 (t1/2 approximately 30 days), whereas the exposed CD4 showed a kinetics of disappearance characterized by two half-life times: a short one (t1/2 approximately 1 day) and a long one approximately equal to that of the RBCs. No side effects or anti-CD4 immune responses were observed in patients over a period of 28 days. The RBC-CD4 entity appears to be long-lived and has no adverse effect in HIV patients.

Adult↗
PubMed1995

Signal transduction in SF9 insect cells: endocytosis of recombinant CD4 after phorbol ester treatment.

In Spodoptera frugiperda (Sf9) insect cells infected with a recombinant baculovirus carrying a gene construct encoding human CD4 endocytosis of CD4 is induced after stimulation with phorbol-12-myristate-13-acetate (PMA). Stimulation of endocytosis with PMA reduced the amount of full-length CD4 on the plasma membrane of Sf9 cells by 50% after 2 hours. Endocytosis of CD4 is blocked after intracellular delivery by cationic liposomes of a monoclonal antibody directed against a cytoplasmic sequence of CD4. Endocytosis is also blocked by the calmodulin inhibitor W7. The PKC inhibitor H7 does not inhibit PMA-induced endocytosis. A truncated CD4, in which the last 32 C-terminal amino acids were deleted did, not respond to PMA. Our results show that PMA can stimulate the calmodulin-dependent signal transduction for endocytosis of full-length CD4 in Sf9 cells. Phosphorylation of CD4 in Sf9 cells was not detectable after PMA treatment and PKC is not required for endocytosis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Stability and immunological reactivity of recombinant membrane CD4 electroinserted into the plasma membrane of erythrocytes.

Concentration-dependent electroinsertion of recombinant human membrane CD4 in human erythrocytes shows a saturation at an average of about 3,500 inserted CD4 epitopes per cell, detectable by flow cytometry. The erythrocyte recovery drops to 10% at this high level of electroinsertion. Experimentally an optimum for cell recovery and insertion rate was found at about 2,500 CD4 epitopes per red blood cell. In vitro stability assay by flow cytometry indicated a temperature- and medium-dependent decrease in the number of CD4 epitopes inserted per cell. This decrease is biphasic, with an exponential part during the first 24 h after electroinsertion followed by a much slower linear decay.

CD4 Antigens↗
PubMed1995

Low-oxygen-affinity red cells produced in a large-volume, continuous-flow electroporation system.

BACKGROUND: Human red cells containing inositol hexaphosphate (IHP) have a lowered O2 affinity, though they are able to bind and carry about the same amount of oxygen as native cells. These modified cells therefore deliver oxygen more efficiently to the tissues, which is a property of potential clinical utility. Investigators set out to devise a system and procedure by which large volumes of IHP-containing red cells, suitable for transfusion, could be produced quickly and efficiently. STUDY DESIGN AND METHODS: The encapsulation of IHP into human red cells by use of several variations of static electroporation was performed to define the conditions necessary for optimal IHP incorporation and cell survival. These conditions were used as a starting point for optimization of a flow electroporation system. RESULTS: When fresh human red cells in a 35 mM IHP solution are subjected to three exponential pulses of field strength of 2.98 +/- 0.064 kV per cm per pulse and pulse length of 2.0 +/- 0.2 msec per pulse while flowing through a cooled electroporation chamber, the condition of the resultant cells, according to the criteria used here, is optimized. After storage for 24 hours in plasma at 37 degrees C, the cells show more than 85-percent survival (in vitro) and hematologic indices nearly identical to those of unpulsed control cells. The p50 value of these cells, however, has doubled to 50.4 +/- 2.0 torr. The processing time for 1 unit of blood is 90 minutes. CONCLUSION: These data indicate that the system described here can efficiently produce low-oxygen-affinity red cells in volumes that are useful in clinical applications.

Electroporation↗
PubMed1995

Specific targeting of human hepatocellular carcinoma cells by immunoliposomes in vitro.

The monoclonal antibody AF-20 was raised against the human hepatocellular carcinoma (HCC) cell line FOCUS and binds with high affinity to a rapidly internalized 180-kd homodimeric glycoprotein that is abundantly expressed on the surface of human HCC and other human cancer cell lines. Immunoliposomes were produced by covalently coupling AF-20 to liposomes containing carboxyfluorescein. Interaction of immunoliposomes with various HCC cell lines in vitro was quantitatively assessed by flow cytometry and qualitatively analyzed by fluorescence microscopy. Liposomes bearing an isotype-matched nonrelevant monoclonal antibody (MAb) and cell lines not expressing AF-20 antigen served as controls. AF-20-immunoliposomes specifically bound to HCC and other human cancer cell lines expressing the AF-20 antigen and were rapidly internalized at 37 degrees C. Interaction of AF-20-conjugated liposomes with these cell lines was between 5 and 200 times greater than that of unconjugated liposomes, whereas no difference was observed between control liposomes bearing a nonrelevant antibody and unconjugated liposomes. Specificity of liposome-target cell interaction was confirmed by competitive inhibition assays. Kinetic analysis showed rapid association of AF-20 immunoliposomes with target cells, with saturation conditions being reached after 60 minutes. We conclude that the MAb AF-20 directs highly efficient, specific, and rapid targeting of immunoliposomes to human HCC and other human cancer cell lines in vitro. This targeted liposomal delivery system represents a promising approach for the development of immunotargeted diagnosis and therapy strategies against HCC.

Antibodies, Monoclonal↗
PubMed1993

Nucleated cells response to protein electroinsertion.

Application of an electrical pulse field at a strength slightly below the value required for electroporation to a suspension of red blood cells in the presence of membrane xenoproteins leads to the insertion of those proteins in the erythrocyte plasma membrane. This observation is extended to nucleated cells. In the presence of glycophorin A, application of such pulses leads to the insertion of 10(4)-10(5) molecules of glycophorin A per cell in CEM-CM3, Hela S3, and bovine CD8+ T cells. Electroinserted glycophorin A is detected by flow cytometry using anti-glycophorin monoclonal antibodies. The survival of the cells subjected to electroinsertion was 55% for CEM-CM3 cells, 69% for Hela S3 cells, and 65% for CD8+ T cells. Cells cultured after electroinsertion lost the electroinserted glycophorin A, with two different rates, by a temperature and cell type-dependent mechanism. During the first 2 h after electroinsertion, the CD8+ T cells lost 12.5% of the inserted glycophorin A per h, the CEM-CM3 cells lost 7.7% per h, whereas the Hela S3 cells lost only 0.8% of the inserted protein per h. After 2 h, the rate increased substantially, to 41.7% per h for the CD8+ T cells, 13.5% for the CEM-CM3 cells, and 8.9% for the Hela S3 cells. Cytochalasin D efficiently inhibited the disappearance of electroinserted glycophorin A during the first 2 h after electroinsertion only.

Animals↗
PubMed1993

Sonographic diagnosis and hemodynamic correlation in veno-occlusive disease of the liver.

This study evaluated the role of duplex Doppler sonography in the diagnosis of VOD of the liver after bone marrow transplantation. Sixteen patients with clinical criteria of VOD were studied. The final diagnosis was achieved by transjugular liver biopsy, and the hepatic venous pressure gradient was measured during the procedure to estimate portal pressure. Nine patients (56.2%) had histologically proved VOD. Gallbladder wall thickening was present in 75% of patients with VOD, but in none of those without it (P = 0.01). Gallbladder wall thickening and ascites were present only in patients with portal hypertension. Nine patients (five with VOD and four without it) also were evaluated with duplex Doppler sonography, and no obvious flow abnormalities were detected in the portal vein and hepatic veins. These results suggest that sonography is useful in detecting early signs of portal hypertension pointing to the diagnosis of VOD in patients with bone marrow transplantation.

Adolescent↗
PubMed1992

Intestinal tuberculosis in AIDS.

The radiological appearance of intestinal tuberculosis (IT) in six patients with AIDS is presented. Involvement of the ileocecal segment (five cases), cecum (four cases), and duodenum (one case) was seen on computed tomographic (CT) or barium studies. There were no significant differences in radiological manifestations of IT among patients with or without AIDS, but all six patients had an advanced stage of their disease at the time of diagnosis.

Acquired Immunodeficiency Syndrome↗