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1.  Class specific immunoglobulin response to individual polypeptides of Chlamydia trachomatis, elementary bodies, and reticulate bodies in patients with chlamydial infection. 
Journal of Clinical Pathology  1986;39(12):1313-1316.
Sera from 10 women with Chlamydia trachomatis culture positive cervicitis and sera from six men with C trachomatis positive non-gonococcal urethritis were studied for the presence of IgG, IgM, and IgA antibodies to polypeptides of C trachomatis elementary bodies and reticulate bodies using immunoblotting techniques. All the sera with IgG, IgM, or IgA immunoglobulins specific to C trachomatis recognised the major outer membrane protein (MOMP) of elementary bodies. IgG antibodies also detected several other proteins, whereas IgM immunoglobulins recognised only MOMP and proteins of 60 kD, 62 kD, and 66 kD. The IgA reacted with MOMP and the 60 kD and 62 kD proteins in elementary bodies. Class specific antibody response against the proteins of reticulate bodies was similar to that observed for elementary body antigens--with one substantial difference: no reaction was observed in the 60 kD and 62 kD positions. This suggests that 60 kD and 62 kD proteins are deficient in reticulate bodies.
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PMCID: PMC1140794  PMID: 3805317
4.  FUNCTIONAL STUDIES OF NEW GLA GENE MUTATIONS LEADING TO CONFORMATIONAL FABRY DISEASE 
Biochimica et biophysica acta  2009;1802(2):247-252.
Fabry Disease (FD) is an X-linked multisystemic lysosomal disorder caused by mutations of α-galactosidase (GLA) gene. Only a few of the 450 genetic lesions identified so far have been characterised by in vitro expression studies. Thus the significance of newly identified GLA nucleotide variants sin FD patients which lead to α-galactosidase (GAL-A) amino acid substitutions or intronic changes can be uncertain.
We identified three GLA mutations: c155G>A (p.C52Y), c548G>C (p.G183A), c647A>G (p.Y216C) in as many individuals (two male; one female), and performed in vitro expression studies and Western Blot analysis, in order to clarify their functional effects.
Reduced GAL-A activity and normal or partially reduced mutant proteins were present in all overexpressed mutant systems in which, three-dimensional structural analysis showed that the active site was not directly involved. We hypothesize that the three new mutations affect the GAL-A protein, leading to conformational FD. When, mutant proteins overexpressed in COS-1 cells and in patients’ lymphocytes were tested in the presence of the 1-deoxygalactonojirimicin (DGJ) chaperone, the p.G183A and p.Y216C systems showed increased GAL-A enzyme activities and protein stabilisation, while p.C52Y was not responsive.
We underline that genetic, biochemical and functional studies are helpful in clarifying the consequences of the missense genetic lesions detected in FD. ERT is the elective therapy for Fabry patients but it is not always possible to issue the enzyme’s active form in all involved organs. Our study endorses the hypothesis that an active-site-specific chemical chaperone, which could be administered orally, might be effective in treating GAL-A conformational defects.
doi:10.1016/j.bbadis.2009.11.003
PMCID: PMC3056268  PMID: 19941952
α-galactosidase; 1-deoxygalactonojirimicin; chaperones; Fabry disease
5.  Serum specific IgA antibody to Chlamydia trachomatis in patients with chlamydial infections detected by ELISA and an immunofluorescence test. 
Journal of Clinical Pathology  1984;37(6):686-691.
Sera obtained from 34 men with Chlamydia trachomatis positive non-gonococcal urethritis, 34 men with C trachomatis negative non-gonococcal urethritis, 42 women with acute salpingitis, 38 healthy women, and 34 healthy men were studied for the presence of specific serum C trachomatis IgA and IgG antibodies. Serological results were correlated with C trachomatis isolation in cell culture. An enzyme linked immunosorbent assay (ELISA) for C trachomatis specific serum IgA was employed using highly purified elementary bodies of C trachomatis serotype L2 grown in LLC-MK2 cells. Results obtained for C trachomatis IgA antibody by the ELISA test were compared with results obtained for the same sera by a single antigen immunofluorescence technique. A good correlation (r = 0.91) was found between two methods. Serum IgG antibody was also determined in the same sera by the immunofluorescence technique. Patients with C trachomatis positive non-gonococcal urethritis had a significantly (p less than 0.0005) higher prevalence (94.1%) of serum IgA antibody by ELISA compared with patients with C trachomatis negative non-gonococcal urethritis (20.5%) or healthy men (5.9%). Similarly, women with acute salpingitis had a significantly (p less than 0.005) higher prevalence of serum IgA antibody (45.2%) compared with healthy controls (5.2%). Comparable results were obtained for C trachomatis serum IgA antibody using the immunofluorescence technique. The prevalence of C trachomatis IgG antibody was significantly higher in patients with C trachomatis positive non-gonococcal urethritis (97.0%) compared with those with C trachomatis negative non-gonococcal urethritis (33.3%) and healthy controls (23.5%). The importance of using specific C trachomatis serum IgA in the identification of chlamydial infection is discussed.
PMCID: PMC498847  PMID: 6373840
6.  The extraperitoneal prosthetic repair of abdominal wall defects in the elderly 
BMC Geriatrics  2010;10(Suppl 1):A6.
doi:10.1186/1471-2318-10-S1-A6
PMCID: PMC3290204
9.  Characterization of a new isolate of Chlamydia trachomatis which lacks the common plasmid and has properties of biovar trachoma. 
Infection and Immunity  1997;65(7):2965-2969.
A Chlamydia trachomatis urethral isolate, alpha/95, yielding pgp3-negative but otherwise normal inclusions by immunofluorescence also gave negative results when pCT-homologous DNA was searched by PCR and Southern blotting. omp-1 sequence analysis identified alpha/95 as a new genotype B variant. These findings confirm that pCT is not required for chlamydial growth in vitro.
PMCID: PMC175415  PMID: 9199473
10.  Detection of serum antibodies to CagA and VacA and of serum neutralizing activity for vacuolating cytotoxin in patients with Helicobacter pylori-induced gastritis. 
Thirty patients with dyspepsia, with histological diagnosis of gastritis, and with endoscopic diagnosis of peptic ulcer disease (PUD) (n = 13) or nonulcer dyspepsia (NUD) (n = 17) were admitted to the study. Helicobacter pylori vacuolating cytotoxin-producing strains (Tox+) were isolated from 14 (46.7%) patients, whereas non-cytotoxin-producing (Tox-) H. pylori strains were isolated from the remaining patients. Of 30 patients studied, 20 (66.7%) had serum cytotoxin neutralizing activity in vitro. Fourteen patients with Tox+ H. pylori strains showed serum cytotoxin neutralizing activity and serum immunoglobulin G (IgG) and IgA antibodies reactive with both 87-kDa H. pylori vacuolating cytotoxin (VacA) and 128-kDa cytotoxin-associated gene product (CagA) by immunoblotting using native enriched preparations of VacA and CagA proteins from H. pylori culture supernatants as the antigens. A 94-kDa antigen cross-reacting with the 87-kDa VacA protein could be demonstrated in culture supernatant with immune sera from humans and animals. All patients (n = 10) lacking serum neutralizing activity were also negative for IgG or IgA against VacA antigen, whereas 6 of the 10 patients showed IgG serum antibody responses against CagA antigen. The prevalence of antibodies to VacA and CagA antigens was significantly (P < 0.001) higher in patients with gastritis (20 and 26 patients for VacA and CagA, respectively, of 30 patients) than in H. pylori culture-negative controls (0 of 27 for both VacA and CagA) and in randomly selected blood donors (17 and 21 for VacA and CagA, respectively, of 120 subjects). All patients with PUD had antibodies to CagA, whereas 13 of 17 (76.5%) patients with NUD had anti-CagA antibodies. Serum IgG antibodies to VacA were present in 9 (69.2%) patients with PUD of 13 patients and in 11 (64.7%) patients with NUD of 17 patients. Anti-CagA antibodies seemed to correlate better with PUD than anti-VacA antibodies.
PMCID: PMC170554  PMID: 9220168
11.  Capture-ELISA for serum IgM antibody to respiratory syncytial virus. 
The Journal of Hygiene  1986;97(3):511-517.
A four-component solid-phase capture enzyme immunoassay was set up to test for serum IgM antibody to respiratory syncytial (RS) virus and was compared with immunofluorescence assay (IFA). A total of 128 young children with acute respiratory infections were studied. Thirty-six were shown to be RS virus-positive by the detection of RS virus in nasopharyngeal secretions and 92 were RS virus-negative. A serum specimen was collected after admission to the hospital (days 0-4) and a further specimen was obtained during days 10-14. Out of 36 RS virus-positive patients, 28 (77.7%) were found to be positive for IgM by both capture-ELISA and IFA. Out of 92 RS virus-negative patients 5 (5.4%) were IgM-positive. Four false-positive results were obtained by IFA due to the presence of rheumatoid factor. The capture-ELISA was shown to be a reliable technique in detecting specific IgM antibody to RS virus.
PMCID: PMC2082896  PMID: 3540115
12.  A rapid immunoperoxidase assay for the detection of specific IgG antibodies to Chlamydia trachomatis. 
Journal of Clinical Pathology  1983;36(3):353-356.
A technique, using indirect immunoperoxidase antibody (IPA), was developed for the detection of IgG antibody to Chlamydia trachomatis. The IPA technique employs glass slides with air-dried and acetone-fixed C trachomatis infected cells, which can be stored at -70 degrees C and used for several months. Antibody titres detected by IPA were comparable to those detected by the indirect fluorescent antibody technique.
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PMCID: PMC498213  PMID: 6338060
13.  Interaction between fibrinogen and cultured endothelial cells. Induction of migration and specific binding. 
It has been suggested that fibrinogen (fg) or its physiological derivatives influence the motility and growth of endothelial cells (ECs), but direct support for this concept is still lacking. In the present study, the capacity of fg to interact with ECs and induce the migration of ECs was examined. The capacity of fg to induce EC migration was studied by means of a modification of the Boyden chamber technique. fg in the lower compartment of the chamber caused a time- and concentration-dependent migration of ECs across filters. fg present in equal concentrations above and below the filter increased EC migration, but the maximal effect invariably occurred in the presence of a gradient between the lower and the upper compartments. Trypsin or plasmin digestion of fg and preincubation of fg with Fab fragments from specific antibody completely abolished fg-induced EC migration. Dialysis of fg to eliminate small peptides that might contaminate the preparation did not modify fg-induced migration. Plasma obtained from healthy donors induced EC migration, but plasma from an afibrinogenemic patient was completely ineffective. The addition of purified fg to afibrinogenemic plasma restored plasma-induced EC migration. Plasmin degradation fragments D and E, of 100,000 and 50,000 mol wt, respectively, did not induce EC migration. However, fragment E caused dose-related inhibition of fg-induced EC migration Direct interaction of highly purified radioiodinated human fg with cultured human and bovine Ecs was observed. The binding was time dependent and plateaued at 10 min. Nonlabeled fg in a large molar excess inhibited the interaction, but unrelated proteins, including fibronectin, ovalbumin, and myoglobin, did not. Monospecific Fab fragments directed to fg inhibited binding by 38% at a 50 to 1 molar ratio whereas nonimmune Fab caused only 2% inhibition at a similar concentration. The binding of 125I-fg with ECs was saturable, and an apparent dissociation constant of 0.23 x 10(-6) M was estimated from binding isotherms. After 30 min of incubation the interaction between 125I-fg and the cells was completely reversible and displaceable by a large molar excess of unlabeled fg. Autoradiography of the display of EC-bound 125I on polyacrylamide gel showed the constitutive B beta- and gamma-chains of the fg molecule, with a partial loss of the A alpha-chain. Purified fragment E and E were tested for their capacity to inhibit fg binding. At a 1 to 400 125I-fg-to-fragment molar ratio, fragment E, which also inhibited migration, competed for binding by 44%, but fragment D was completely ineffective. These data show that fg may specifically associate with ECs and induce migration of these cells; it also appears that the structural requirement of this activity is located in the N-terminal part of the molecule.
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PMCID: PMC423387  PMID: 3965498
14.  An immunoperoxidase assay for the detection of specific IgA antibody in Epstein-Barr virus infections. 
Journal of Clinical Pathology  1984;37(4):440-443.
A technique using indirect immunoperoxidase antibody was developed for the detection of specific serum IgA antibody to Epstein-Barr virus capsid antigen and early antigen. The IgA technique was compared with an immunofluorescence antibody method. Epstein-Barr virus IgA antibody against viral capsid antigen was detected in all nine patients with Epstein-Barr virus associated undifferentiated nasopharyngeal carcinoma, in 13 (72.2%) of 18 patients with infectious mononucleosis, in 21 (28.3%) of 74 patients with acute lymphoblastic leukaemia, and in six (20%) of 30 patients who had recently had kidney transplants. Epstein-Barr virus IgA antibody against viral capsid antigen was also detected in four (10%) of 40 healthy subjects, but it was not found in any of 20 cord blood samples. Epstein-Barr virus IgA antibody to early antigen was detected in six (66.6%) patients with nasopharyngeal carcinoma and in two (2.7%) patients with acute lymphoblastic leukaemia. The immunoperoxidase assay for Epstein-Barr virus specific IgA was simple, reliable, and rapid and correlated well (r = 0.94) with the immunofluorescence antibody technique.
PMCID: PMC498747  PMID: 6323549
15.  Rapid immunoperoxidase assay for detection of respiratory syncytial virus in nasopharyngeal secretions. 
Journal of Clinical Microbiology  1983;18(4):947-949.
Samples of nasopharyngeal secretions obtained from 70 infants and young children with acute respiratory disease were examined for the presence of respiratory syncytial virus by immunoperoxidase assay (IPA). The IPA was compared with the immunofluorescence assay and with cell culture isolation. Respiratory syncytial virus antigen-positive cells were detected by both IPA and immunofluorescence assay in 28 specimens; 25 samples were positive in cell culture. The agreement between virus isolation and IPA and IFA was 89%. The applicability of IPA to rapid viral diagnosis of respiratory syncytial virus infection is discussed.
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PMCID: PMC270936  PMID: 6355168
16.  Cultured human endothelial cells generate tissue factor in response to endotoxin. 
Journal of Clinical Investigation  1983;71(6):1893-1896.
Bacterial infection is associated with disseminated intravascular coagulation and fibrin deposition in the microcirculation; the mechanism of these effects in humans is still unclear. We have studied the generation of procoagulant activity (PCA) by cultured human endothelial cells (EC) in response to endotoxin. Cells from umbilical cord veins were grown in Eagle's minimum essential medium with 20% fetal calf serum till confluence. Absence of fibroblasts and macrophages was carefully checked. Endotoxin (Salmonella enteritidis lipopolysaccharide (LPS) W or Escherichia coli 0111:B4 LPS W, 0.01-1.0 micrograms/ml) was added to culture dishes for 4-6 h. PCA of EC was measured by a one-stage clotting assay and/or a two-stage amidolytic assay with the chromogenic substrate S-2222. In the absence of endotoxin, EC generated little, if any PCA (2-5 units/10(5) cells). In contrast, the addition of endotoxin resulted in generation of strong PCA that reached a maximum within 4-6 h (185-241 units/10(5) cells) and was dose-dependent between 1 and 0.01 microgram endotoxin/ml of culture medium. The generation of PCA required RNA and protein synthesis but did not require the presence of serum. No activity was found in the culture medium. The activity was of tissue thromboplastin type, as indicated by biological and immunological criteria. These endotoxin effects were observed in the absence of endothelial damage, as shown by phase-contrast microscopy and lack of 51Cr release. These data could contribute to elucidate the pathogenesis of vascular complications associated with endotoxemia in man.
PMCID: PMC370395  PMID: 6345590
18.  Cytological and histopathological abnormalities of the cervix in genital Chlamydia trachomatis infections 
Since genital infection with Chlamydia trachomatis may be associated with cervical abnormalities 160 patients with grandular ectopia attending a gynaecological outpatient clinic were examined for antibodies against C trachomatis, the presence of C trachomatis infection, and cytological and histopathological abnormalities of the cervix.
A significantly higher incidence of histological dysplasia was found in women with glandular ectopia who had antichlamydial antibodies than in those without.
PMCID: PMC1045959  PMID: 7296254
19.  Plasmatic regulation of vascular prostacyclin in pregnancy. 
Activity of prostacyclin-stimulating factor was measured in six normal, non-pregnant women, six women in early normal pregnancy, six in late normal pregnancy, and six in late pregnancy complicated by severe pre-eclampsia. The activity was lower in the women in late pregnancy than in those in early pregnancy and the controls but was about normal in those with severe pre-eclampsia. These results may be relevant to the physiology of pregnancy and the pathogenesis of pre-eclampsia.
PMCID: PMC1504327  PMID: 6780103
20.  Tumour sublines with different metastatic capacity induce similar blood coagulation changes in the host. 
British Journal of Cancer  1981;43(1):100-104.
This paper is aimed at investigating how metastatic tumour growth influenced the haemostatic system of the host. Blood platelet count, blood fibrinogen level, the activated partial thromboplastin time (APTT) and the prothrombin time (PT) were determined at various intervals during growth and metastasis of a murine fibrosarcoma (mFS6) or one of its sublines with different metastatic capacity. Progressive thrombocytopenia and increase in fibrinogen level were observed during development of the tumour in all the animal groups studied, irrespective of the metastatic potential of the various sublines. No significant changes were observed in the PT or APTT values. These data support the concept that primary rather than metastatic growth influences the haemostatic system of tumour-bearing animals.
PMCID: PMC2010497  PMID: 7459231
22.  Intermittent heparin treatment does not induce hypercoagulability in haemodialysed patients. 
Journal of Clinical Pathology  1980;33(7):631-634.
Antithrombin-III (AT-III) and factor VIII coagulant (F VIII:C) and antigenic (F VIII:RA) activities have been studied in nine conservatively treated and 26 dialysed uraemic patients. AT-III levels were not significantly different from those of controls in either group. Among dialysed patients, those who had experienced thrombotic occlusions of the vascular accesses could not be distinguished from the remaining patients on the basis of their AT-III levels. Both F VIII:C and F VIII:RA were slightly higher than in controls in conservatively treated patients, but significantly higher in haemodialysed patients, especially in those who had never experienced thrombotic complications of the vascular accesses. No acute changes were observed in either the AT-III or F VIII:C/F VIII:RA ratio in five patients given heparin therapy during a dialytic session or in the interdialytic period. Thus repeated intermittent heparin treatment does not induce a hypercoagulable state in haemodialysed patients.
PMCID: PMC1146174  PMID: 6776154
23.  Oral contraceptives and the prothrombin time. 
British Medical Journal  1980;280(6210):332-333.
PMCID: PMC1600131  PMID: 7357363
24.  Evidence that cells from experimental tumours can activate coagulation factor X. 
British Journal of Cancer  1979;40(2):228-233.
The procoagulant activity of cells from some experimental tumours isolated in culture or in single-cell suspensions from ascitic fluid was investigated. Cells from Lewis lung carcinoma (primary and metastasis), Ehrlich carcinoma ascites and JW sarcoma ascites were able to shorten markedly the recalcification time of normal, Factor VIII- and Factor VII-deficient but not of Factor X-deficient human plasma. The same cells generated thrombin when mixed with a source of prothrombin and Factor X, absorbed bovine serum (as a source of Factor V), phospholipid and calcium chloride. Thrombin formation was not influenced by the presence of Factor VII. Cells from Sarcoma 180 ascites were completely inactive in both test systems. It is concluded that cells from some experimental tumours have the capacity to activate Coagulation Factor X directly. These findings suggest the existence of an alternative "cellular" pathway in the initiation of blood clotting distinct from both the intrinsic and extrinsic mechanisms.
PMCID: PMC2010022  PMID: 573131
25.  Factor VIII--related protein on vascular intima of patients with chronic renal failure and prolonged bleeding times. 
British Medical Journal  1978;1(6105):70-72.
To determine whether the prolonged bleeding time so common to chronic renal failure (CRF) was due to defective factor VIII-related activities, as in von Willebrand's disease, vascular-factor VIII-related protein was measured in patients with CRF. Factor VIII-related protein was detected by immunofluorescence on the vascular intima of all 13 patients with CRF and greatly prolonged bleeding times. This protein was also present on the vascular intima of a patient with CRF and moderate von Willebrand's disease. These findings support a previous suggestion that the disturbed haemostasis in patients with CRF is not linked to defective factor VIII-related activities.
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PMCID: PMC1602594  PMID: 339992

Results 1-25 (28)