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Biomedical subjects

J Ingerslev

Publications and source records attributed to J Ingerslev.

At least 127 records · Page 7Linked to original sources
PubMed1989

Female haemophilia A in a family with seeming extreme bidirectional lyonization tendency: abnormal premature X-chromosome inactivation?

We studied a female child with mild classical haemophilia A, presenting with a F VIII deficiency similar to that detected in her maternal grandfather. Investigations on several occasions showed that the obligate carrier mother of the proposita had normal VIII:C activity, whereas her likewise obligate carrier sister had a typical carrier VIII:C/vWf:Ag pattern. The child was a phenotypically normal female with normal karyotype. Her father had no clinical or biochemical signs of haemophilia A. RFLP-analysis using DX13 and St14 probes each elicited one allele (5.8 and 3.4 kb, respectively) segregating along with the affected F VIII gene from the hemizygous grandfather to both his daughters and further to the haemophilic female child. The paternity of the child was analyzed using various red cell and HLA antigens and RFLP by p29C, a probe detecting polymorphic hypervariable TaqI and PstI fragments in the pseudoautosomal areas of the X- and Y-chromosomes. All results obtained were concordant with the declared paternity. RFLP-analysis, using single (Pst I) and double digestion (Pst I/Hha I) of DNA and a PGK probe, revealed a remarkable difference in hybridization fragments, strongly suggesting hypermethylation, and in consequence, preferential X-chromosome inactivation in the proposita. This points to extreme lyonization as the most plausible explanation for haemophilia A in this female child. A familial tendency to abnormal premature X-chromosome inactivation is speculated.

Child, Preschool↗
PubMed1988

Synthesis of factor VIII in human hepatocytes in culture.

Although several investigators have attempted to identify the site of synthesis of factor VIII (FVIII), the cellular species responsible for maintenance of plasma FVIII has not been clearly defined. Indications point at hepatocytes and certain endothelial cells. The present study investigated the FVIII coagulant antigen (VIII:Ag) of hepatocytes obtained by two-step collagenase digests of human liver pieces. Following Percoll gradient centrifugation, less than 1% of cells harvested were non-parenchymal. Lysates of freshly isolated and purified hepatocytes contained 165-250 mU of VIII:Ag/10(6) cells as defined by a two-site ELISA employing a haemophilic antibody against human FVIII. This material contained a single peak of VIII:Ag polypeptides as judged from the VIII:Ag ELISA profile of Mono-Q fast protein liquid chromatography fractions. A haemophilic antibody specific for epitopes of the light chain of FVIII, employed in immunoisolation of VIII:Ag in lysate of human hepatocytes, extracted a polypeptide pattern that was studied in a reduced SDS-PAGE electrophoresis gel and compared to that of immunoisolate from normal plasma. After electroblotting onto nitrocellulose and reaction with a monoclonal antibody towards the light chain of FVIII, the appearance of a doublet at 78-79 kDa in both these materials indicated the presence of the light chain of FVIII in human hepatocyte lysate. During culture, human hepatocytes secreted 20-80 mU of VIII:Ag per 1 x 10(6) cells per 24 hours. Further, a significant secretion of VIII:Ag was found in media of cultured human hepatoma cells, Hep-G2, whereas human blood monocytes and human fibroblasts did not secrete detectable VIII:Ag. In all of these cell cultures, vWf:Ag was indetectable or present as trace.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1988

Applications of immunoperoxidase techniques in specificity testing of monoclonal antibodies (Mabs) against von Willebrand factor (vWf).

The present communication describes the production of a new series of murine Mabs against von Willebrand factor (vWf) in which specificity was tested using immunoperoxidase techniques. Seven Mabs showed specific reactivity with native and disaggregated vWf, whereas no binding was found to material from patients with severe homozygous (or doubly heterozygous) von Willebrand's disease (vWd) or factor VIII coagulant antigen (VIII:Ag). These Mabs are thought to carry separate specificities as only slight or no competitive activity was detected. Four Mabs partially inhibited the ristocetin-induced platelet agglutination and three interacted with vWf-binding to type I collagen. All antibodies bound to the complete range of vWf multimers of normal plasma. Excellent binding and detection properties of Mabs were found in asymmetrical two-site enzyme linked immunosorbent assays (ELISA) for quantitation of vWf antigen (vWf:Ag). One particular antibody (Mab vWf-33) discriminated vWf material from a number of subtype II vWd plasmas tested.

Animals↗
PubMed1988

Kidney tissue somatomedin C and initial renal growth in diabetic and uninephrectomized rats.

Kidney growth after induction of experimental diabetes in rats was compared to compensatory renal growth in response to unilateral nephrectomy. After 4 days of diabetes, kidney weight had increased from 816 +/- 21 mg (SEM) to 940 +/- 42 mg (15%). In insulin-treated diabetic rats kidney weight was unchanged at the end of the study, namely 828 +/- 15 mg. In unilaterally nephrectomised rats kidney weight increased from 840 +/- 20 mg (SEM) to 1050 +/- 60 mg during 4 days (24%). We observed increased kidney content of somatomedin C in both diabetic and uninephrectomized rats. In untreated diabetic rats it was maximal after 48 h, with an increase of 77% (3469 +/- 312 ng/g (SEM) versus 1961 +/- 173 ng/g). After 4 days the somatomedin C content had returned to initial levels. In insulin-treated rats somatomedin C content did not increase during the observation period. The somatomedin C content of the remaining kidney after unilateral nephrectomy was maximal after 24 h with an increase of 58% (from 1340 +/- 203 ng/g (SEM) to 2122 +/- 214 ng/g). The somatomedin C content returned to normal at day 4. Serum somatomedin C declined insignificantly in diabetic animals during the experimental period, but a significant decrease (p less than 0.02) was found in uninephrectomized rats. This study demonstrates that kidney somatomedin C peaks during the first or second day after uninephrectomy or induction of diabetes, respectively, and that insulin treatment sufficient to prevent kidney growth abolishes the increase. These similar rapid initial hypertrophies/hyperplasies may thus be dependent on local somatomedin C formation.

Animals↗
PubMed1988

Synthesis and secretion of alpha 2-macroglobulin by human hepatocytes in culture.

Hepatocytes were isolated by application of the two-step collagenase perfusion technique to pieces of human liver. The cells were incubated in serum-free medium or 10% FCS-medium supplemented with insulin, glucagon and dexamethasone, and kept in culture for more than 2 weeks. Seventy-five per cent of the medium was changed regularly and assayed for alpha 2-macroglobulin (alpha 2-M), pregnancy zone protein, alpha 1-antitrypsin and albumin by means of ELISA. Significant amounts of alpha 2-macroglobulin were present in all cultures. During incubation, alpha 2-M accumulated in the medium and the quantity of alpha 2-M released from the cells by far exceeded protein associated with hepatocytes prior to incubation. In 24 h 10(6) hepatocytes secreted 160.5 +/- 82.2 ng of alpha 2-M (mean +/- SD, n = 5). Cell-associated, as well as secreted alpha 2-M appeared to be on native form, as determined by immunoisolates from lysed cells and culture supernatants. Pregnancy zone protein was only detected in about 50% of the cultures and its rate of secretion was less than 2 ng 24 h-1 per 10(6) cells. In contrast, culture medium contained considerable quantities of alpha 1-antitrypsin and albumin. In 24 h, 10(6) hepatocytes released greater than 2 micrograms alpha 1-antitrypsin and greater than 5 micrograms albumin. The present study suggests the hepatocyte to be of major importance for the synthesis of intravascular alpha 2-M.

Adult↗
PubMed1988

Human hepatocytes exhibit receptors for alpha 2-macroglobulin and pregnancy zone protein-proteinase complexes.

Hepatocytes were isolated by application of the two-step collagenase technique to pieces of human liver. 125I-labelled alpha 2-macroglobulin-trypsin complex bound to hepatocytes at 4 degrees C with a half time of approximately 4.5 h. At near equilibrium half of the receptors were saturated at an alpha 2-macroglobulin-trypsin complex concentration of about 60 pmol 1(-1) and the Scatchard plot was linear. Dissociation of the labelled complex was slow (T1/2 = 24 h) at low receptor occupancies. At high receptor occupancies dissociation was biphasic with a rate constant (K-1) for the initial rapid phase of about 2.4 x 10(-2) min-1. Labelled alpha 2-macroglobulin-trypsin complex bound at 4 degrees C was rapidly internalized at 37 degrees C (T1/2 = 1.9 min), and in 3.5 h approximately 10% of the label was released into the medium in a trichloroacetic acid-soluble form. At 37 degrees C, 125I alpha 2-macroglobulin-trypsin was taken up by hepatocytes and trichloroacetic acid soluble radioactivity appeared in the medium following a sigmoidal curve. Similar results were obtained with 125I-pregnancy zone protein-chymotrypsin complex. At 4 degrees C, hepatocytes bound nearly equal amounts of labelled alpha 2-macroglobulin-trypsin and pregnancy zone protein-chymotrypsin complex, and a large excess (100 nmol 1(-1) of one of the macroglobulins could almost completely abolish binding of trace amounts (5-20 pmol 1(-1] of the other. The present findings strongly suggest that the hepatocyte is of major importance for removal of alpha 2-macroglobulin- and pregnancy zone protein-proteinase complex in humans, in agreement with previous results in rats and mice.

Biological Transport, Active↗
PubMed1988

Pattern of immunoglobulin classes and IgG subclasses of platelet-associated immunoglobulin in HIV-seropositive haemophiliacs.

Abnormal amounts of platelet-associated immunoglobulins (PAIg) and seropositivity for HIV were demonstrated in 18 haemophiliacs suffering from thrombocytopenia of varying severity. Among 34 non-thrombocytopenic haemophiliacs seropositivity for HIV was found in association with increased levels of PAIg in 18 patients, whereas increased amounts of PAIg were seen only in 4 of the 16 patients seronegative for HIV. In most of the haemophiliacs with thrombocytopenia, the PAIg represented all classes of immunoglobulins and in half of the cases all subclasses of IgG, while in 33 patients with autoimmune thrombocytopenic purpura (AITP), PAIg was IgG1 and IgM in most cases. Our data show a difference in the pattern of PAIg in AITP patients and in thrombocytopenic haemophiliacs seropositive for HIV. We suggest that the thrombocytopenia in haemophiliacs is related to the polyclonal B cell activation also found in other patients seropositive for HIV and perhaps indicative of AIDS-related complex.

Blood Platelets↗
PubMed1988

Surgical treatment of severe periodontitis in a haemophilic patient with inhibitors to factor VIII. Report of a case.

This report describes the surgical treatment of advanced periodontitis in a haemophilic patient with inhibitors to Factor VIII. The treatment was performed after substitution therapy with Factor VIII-concentrate, supported by local and systemic antifibrinolytic treatment with tranexamic acid. No complications developed postoperatively, and after 9 months, the patient did not show recurrence of periodontal disease. Although the present case shows, that even severe periodontitis can be treated surgically in haemophilic patients with inhibitors to Factor VIII, this should not be done unless it is absolutely necessary. The treatment of periodontal disease in such patients should be instituted as early as possible in order to prevent the need for extensive surgery or dental extractions.

Antibodies↗
PubMed1988

Rapid and inexpensive microdetermination of serum fructosamine results in diabetics, uraemics, diabetics with uraemia and healthy subjects.

This paper describes a simple, reliable and inexpensive method for rapid determination of serum fructosamine. The assay is based on commercially available reagents and utilizes equipment accessible in most laboratories (i.e. an automated ELISA-reader interfaced with an IBM computer). In contrast to HbA1c determination, the fructosamine method presented can be used in diabetes complicated by uraemia. In the clinically relevant measuring range, fructosamine is uninfluenced by serum albumin concentration in diabetics with or without uraemia. Eighty microlitres of non-haemolysed capillary serum suffices for a duplicate determination. One year's storage of normal serum induced no change in serum fructosamine estimates. s-Fructosamine in 18 healthy subjects was 2.1 +/- 0.3 mmol/l (mean +/- SD) in venous blood and 2.2 +/- 0.4 mmol/l in capillary blood. In diabetics 3.4 +/- 0.6 mmol/l and 3.3 +/- 0.6 mmol/l (n = 38) were found. The method is well-suited for routine use in the diabetic out-patient clinic.

Adult↗
PubMed1988

Silent hepatitis-B immunization in laboratory technicians.

The present investigation was initiated to assess the demand for vaccination against hepatitis-B in a department of clinical chemistry. One hundred and twenty-eight employees enrolled for the test programme consisting primarily of screening for hepatitis-B antigen (HBsAg) and antibody to hepatitis-B surface antigen (anti-HBs). Surprisingly it was found, that 8 persons out of 58 who had also participated in a screening survey in 1978 had developed anti-HBs. These persons, who had no history of hepatitis, were also positive for antibodies towards the core antigen of hepatitis-B (anti-HBc). A seroconversion rate of 1,53/100/year for this material was calculated. Six persons who had contracted hepatitis-B during an epidemic 14 years previously, presented with very weak anti-HBs reactivity, but were positive for anti-HBc indicating, that anti-HBc was the more stable indicator of past HBV infection. The cost-effectiveness of vaccination against HBV seems disputable compared with other protective measures. The high incidence of silent seroconversion indicates a chronic exposure to virus among the laboratory personnel. Therefore, safety precautions against virus exposure are highly recommended.

Adult↗
PubMed1988

Human hepatocytes in culture synthesize and secrete fibronectin.

Hepatocytes were isolated by application of the two-step collagenase perfusion technique to pieces of human liver. Hepatocytes were cultivated in serum-free medium or 10% fetal calf serum medium supplemented with insulin, glucagon and dexamethasone. The cells were kept in culture for up to 16 days and 75% of the medium was regularly changed. Fibronectin in culture medium was detected by means of an ELISA with an assay range of 2.2-560 micrograms/l. The interassay imprecision was 6.3% at 500 micrograms/l and 14.3% at 10 micrograms/l. Significant amounts of fibronectin were detected in all cultures. During culture, fibronectin accumulated in the medium and the quantity secreted by hepatocytes by far exceeded the amounts of fibronectin associated with hepatocytes prior to cultivation. Maximum secretion rate by 10(6) hepatocytes was 167.5 +/- 73.3 ng fibronectin (mean +/- SEM, n = 3) in 24 h. When analysed by means of SDS-PAGE and immunoblotting the fibronectin isolated from hepatocyte culture medium and cell lysate co-migrated with fibronectin obtained from plasma. Our data show, for the first time, that human hepatocytes synthesize and secrete fibronectin, and it is suggested that the human liver is an important source of plasma fibronectin.

Cells, Cultured↗
PubMed1987

Continuous high-dose factor VIII for the induction of immune tolerance in haemophilia A patients with high responder state: a description of eleven patients treated.

Eleven severely affected haemophilia A patients (aged 6-42 y) with F VIII:C inhibitor (high responders) were treated with high-dose F VIII in order to eliminate the inhibitors. The patients comprise Danish high responder patients treated during the period 1977-1985. In all patients the inhibitors decreased significantly. In six, the inhibitor apparently disappeared (detection limit 0.4 Bethesda Units per ml) (BU/ml), in four patients a low level inhibitor of 0.4-1.4 BU/ml persisted. One patient is still on high-dose schedule. The duration of high-dose treatments ranged from less than one month up to 18 months. In all patients the tendency to spontaneous bleedings vanished when a measurable VIII:C level appeared in the post-infusion sample. The inhibitor suppression has allowed for extensive physical training and rehabilitation orthopaedic surgery. The patients are now able to conduct a normal haemophilic life on self-administered prophylactic doses of F VIII.

Adolescent↗
PubMed1987

Characteristics of the von Willebrand factor in virus inactivated F VIII concentrates: the impact of heat treatment.

The known transmission of viral diseases, particularly AIDS (HIV, LAV, HTLV-III), has led to the mandatory use of virus-inactivated coagulation factor concentrates for treatment of bleeding disorders due to deficient or abnormal synthesis of the factor VIII/von Willebrand factor complex. The present investigation was undertaken to study the influence of heat-treatment on the von Willebrand factor (vWf). Using normal plasma as reference material, we studied the influence of low-purification steps in a simple cryo-plasma and a unrefined freeze-dried cryoprecipitate. For comparison, non-heated and heat-inactivated concentrates of different manufacture representing varying heat-treatment protocols were studied using quantitation of von Willebrand factor antigen (vWf:Ag) by electroimmunoassay and ELISA, and investigation of vWf multimeric composition. A locally produced factor VIII concentrate was studied before and after exposure to 68 degrees C for 72 hours (dry state). Whenever possible, commercial preparations manufactured prior to the heat-treatment era were compared with the present product. The locally produced high purity concentrate elicited only minor changes in oligomeric satellite pattern, which did not change after dry heat exposure. In principle, no major differences were found between non-heated and pasteurized commercial concentrates of same manufactural origin.

Antigens↗