Mapping of an autosomal dominant gene for Dupuytren's contracture to chromosome 16q in a Swedish family

Mapping of an autosomal dominant gene for Dupuytren's contracture to chromosome 16q in a Swedish family.Dupuytren's contracture (DC) (OMIM 126900) is the most common connective tissue disease of mankind and has both heritable and sporadic forms. The inherited form is most frequently observed among the xanthochroi peoples of Northern Europe where its most common manifestations are thickening of the palmar fascia and contracture of the fingers. We ascertained a five-generation Swedish family in which DC is inherited in an autosomal dominant manner with high, but incomplete, penetrance by the end of the fifth decade. Blood was collected from all affected and informative unaffected family members for the performance of a genome-wide scan at a resolution of approximately 8 cM for all autosomes. Linkage was established to a single 6 cM region between markers D16S419 and D16S3032 on chromosome 16. A maximal two-point logarithm of odds (LOD) score of 3.18 was achieved at microsatellite marker D16S415 with four other markers in the region producing LODs of >1.5.     

Cryopreservation of single human spermatozoa

A procedure is described that allows cryopreservation and efficient post-thaw recovery of either a single or a small group of human spermatozoa. This is achieved by injecting them into cell-free human, mouse or hamster zonae pellucidae before the addition of cryoprotectant. The method involves a combination of physical micromanipulation procedures and glycerol-mediated cryoprotection. Zonae were tracked by positioning them in straws between two small air bubbles prior to freezing. Spermatozoa from poor specimens were cryopreserved and their fertilizing ability after thawing was compared with that of fresh spermatozoa from fertile men. Human eggs used for fertilization testing were either 1 day old or in-vitro matured. Only 2% of the frozen zonae were lost and >75% of spermatozoa cryopreserved in this manner were recovered and prepared for intracytoplasmic sperm injection. The feasibility of cryopreserving a single spermatozoon was assessed. Fifteen motile spermatozoa were frozen in 15 zonae, of which 14 were recovered after thawing. Ten were injected into spare eggs, of which eight became fertilized. Spermatozoa recovered mechanically from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa. The recovery and fertilization rates with spermatozoa frozen in animal zonae were 87 and 78% respectively. The fertilization rate was marginally higher (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps because the latter may have acrosome reacted more frequently. The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa.      

Detection of yellow fever virus in serum by enzyme immunoassay

Yellow fever (YF) virus is present in patient's blood during the acute phase of illness. Virus isolation and identification provide a potential method of early diagnosis, but available techniques are slow and require specialized materials and equipment. An alternative approach is direct detection of YF antigen in serum by means of an enzyme-linked immunosorbent assay (ELISA). An antigen-capture ELISA was developed, which used anti-YF antibodies, immobilized on a solid phase (polystyrene plates), to capture YF virus from serum samples. After addition of the virus-containing sample, anti-YF detecting antibody conjugated to alkaline phosphatase was added to detect viral antigen. Trials with various capture and detecting antibodies in systems employing purified YF 17D virus, led to the selection of: 1) two capture antibodies (pooled human serum containing high titer YF IgM antibodies and a type-specific YF monoclonal antibody), and 2) a detecting antibody conjugate consisting of monoclonal antibody broadly cross-reactive with all flaviviruses, purified by affinity chromatography, and conjugated to alkaline phosphatase. The limit of sensitivity in tests against purified YF 17D virus diluted in buffer or normal human serum was 10(3.0) - 10(3.6) PFU/0.05 ml or 0.007-0.029 microgram viral protein/0.05 ml. Sera obtained at intervals from rhesus and cynomolgus monkeys after infection with a wild YF virus strain were tested. The limit of sensitivity of the assay applied to viremic monkey serum was similar (approximately 3.5 log10PFU/0.05 ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ill or just old? Towards a conceptual framework of the relation between ageing and disease

Background

Is this person ill or just old? This question reflects the pondering mind of a doctor while interpreting the complaints of an elderly person who seeks his help. Many doctors think that ageing is a non-disease. Accordingly, various attempts have been undertaken to separate pathological ageing from normal ageing. However, the existence of a normal ageing process distinct from the pathological processes causing disease later in life can be questioned.

Discussion

Ageing is the accumulation of damage to somatic cells, leading to cellular dysfunction, and culminates in organ dysfunction and an increased vulnerability to death. Analogously, chronic diseases initiate early in life and their development is slow before they become clinically apparent and culminate in disability or death. The definition of disease is also subject to current opinions and scientific understanding and usually, it is an act of individual creativity when physical changes are recognised as symptoms of a new disease. New diseases, however, are only rarely really new. Most new diseases have gone undiagnosed because their signs and symptoms escaped recognition or were interpreted otherwise. Many physical changes in the elderly that are not yet recognised as a disease are thus ascribed to normal ageing. Therefore, the distinction between normal ageing and disease late in life seems in large part arbitrary.

Summary

We think that normal ageing cannot be separated from pathological processes causing disease later in life, and we propose that the distinction is avoided.

     

Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein

Partially purified preparations of porcine factor VIII:C were used to immunize mice and spleen cells from the immunized animals were fused to NS-1 mouse myeloma cells. The ability of hybrid culture fluids to bind factor VIII:C was detected with a radiolabelled, affinity-purified, human antihuman VIII:C inhibitor. Three cloned hybrid lines have been obtained that preferentially bind to VIII:C when compared to von Willebrand factor binding. Two of these monoclonal antibodies partially inhibit VIII:C coagulant activity. The third antibody does not inhibit VIII:C, but it can be used as an affinity reagent to absorb dissociated VIII:C out of solution. Active coagulant can be recovered by elution in 50% ethylene glycol. The VIII:C obtained has a specific activity of 6 units/micrograms based on absorbance measurements. When analyzed on SDS gels, the unactivated VIII:C contains 3 bands of apparent molecular weight 166,000, 130,000 and 76,000. Thrombin treatment results in a 40 fold increase in activity and cleavage to products of 76,000, 67,000 an 50,000 and small amounts of lower molecular weight peptides. EDTA inactivation of the factor VIII:C results in the separation of the 166,000 and 130,000 chains from the 76,000 chain, suggesting a Ca++ dependent noncovalent interaction among the chains.     

Inactivation of Friend erythroleukemia virus and Friend virus- transformed cells by merocyanine 540-mediated photosensitization

The Friend virus complex was used as a model to study the effects of merocyanine 540 (MC 540)-mediated photosensitization on enveloped viruses. Simultaneous exposure to the lipophilic dye MC 540 and white light inactivated cell-free virus, cell-associated virus, and virus- transformed cells. When used under experimental conditions that are known to preserve most mature blood cells, at least some coagulation factors, and a significant portion of the pluripotent hematopoietic stem cell compartment, MC 540-mediated photosensitization reduced virus titers by greater than or equal to 4 log and the concentration of in vitro clonogenic erythroleukemia cells by greater than or equal to 5 log. Animals that received a single intravenous injection of photosensitized virus were resistant to a subsequent challenge with live virus. High sensitivity to MC 540-mediated photosensitization appears to be a property that is shared by other enveloped viruses. Thus, photosensitization mediated by MC 540 may be of benefit in the sterilization of blood products (in particular, cellular products), the production of vaccines, and selected areas of antiviral therapy.     

Oral sodium phenylbutyrate therapy in homozygous beta thalassemia: a clinical trial

Butyrate analogues have been shown to increase fetal hemoglobin (HbF) production in vitro and in vivo. Sodium phenylbutyrate (SPB), an oral agent used to treat individuals with urea-cycle disorders, has been shown to increase HbF in nonanemic individuals and in individuals with sickle cell disease. We have treated eleven patients with homozygous beta thalassemia (three transfusion dependent) and one sickle-beta- thalassemia patient with 20 g/d (forty 500-mg tablets) of SPB for 41 to 460 days. All patients showed an increase in the percent of F reticulocytes associated with treatment, but only four patients responded by increasing their Hb levels by greater than 1 g/dL (mean increase, 2.1 g/dL; range, 1.2 to 2.8 g/dL). None of the transfusion- dependent thalassemia subjects responded. Increase in Hb was associated with an increase in red blood cell number (mean increase, 0.62 x 10(12)/L), and mean corpuscular volume (mean increase, 6 fL). Changes in percent HbF, absolute HbF levels, or alpha- to non-alpha-globin ratios as measured by levels of mRNA and globin protein in peripheral blood did not correlate with response to treatment. Response to treatment was not associated with the type of beta-globin mutation, but baseline erythropoietin levels of greater than 120 mU/mL was seen in all responders and only two of eight nonresponders to SPB. Compliance with treatment was greater than 90% as measured by pill counts. Side effects of the drug included weight gain and/or edema caused by increase salt load in 2/12, transient epigastric discomfort in 7/12, and abnormal body odor in 3/12 subjects. Two splenectomized patients who were not on prophylactic antibiotics developed sepsis while on treatment. We conclude that SPB increases Hb in some patients with thalassemia, but the precise mechanism of action is unknown.     

Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization

Mice lacking granulocyte colony-stimulating factor (G-CSF) were generated by targeted disruption of the G-CSF gene in embryonal stem cells. G-CSF-deficient mice (genotype G-CSF-/-) are viable, fertile, and superficially healthy, but have a chronic neutropenia. Peripheral blood neutrophil levels were 20% to 30% of wild-type mice (genotype G- CSF+/+) and mice heterozygous for the null mutation had intermediate neutrophil levels, suggesting a gene-dosage effect. In the marrow of G- CSF-/- mice, granulopoietic precursor cells were reduced by 50% and there were reduced levels of granulocyte, macrophage, and blast progenitor cells. Despite G-CSF deficiency, mature neutrophils were still present in the blood and marrow, indicating that other factors can support neutrophil production in vivo. G-CSF-/- mice had reduced numbers of neutrophils available for rapid mobilization into the circulation by a single dose of G-CSF. G-CSF administration reversed the granulopoietic defect of G-CSF-/- mice. One day of G-CSF administration to G-CSF-/- mice elevated circulating neutrophil levels to normal, and after 4 days of G-CSF administration, G-CSF+/+ and G-CSF- /- marrows were morphologically indistinguishable. G-CSF-/- mice had a markedly impaired ability to control infection with Listeria monocytogenes, with diminished neutrophil and delayed monocyte increases in the blood and reduced infection-driven granulopoiesis. Collectively, these observations indicate that G-CSF is indispensible for maintaining the normal quantitative balance of neutrophil production during "steady-state" granulopoiesis in vivo and also implicate G-CSF in "emergency" granulopoiesis during infections.