Identification of a novel region of the GABA(B2) C-terminus that regulates surface expression and neuronal targeting of the GABA(B) receptor.

GABA(B) is a G protein-coupled receptor composed of two subunits, GABA(B1) and GABA(B2). GABA(B1) contains an endoplasmic reticulum-retention sequence and is trafficked to the cell surface only in association with GABA(B2). To determine whether the C-terminus of GABA(B2) regulates GABA(B) trafficking, we constructed forms of GABA(B2) with various C-terminal truncations and examined their surface expression. Truncation of GABA(B2) after residue 841 significantly reduced surface expression of both the subunit and the heterodimerized receptor. Turnover of the Delta841 construct, however, did not differ from that of full-length GABA(B2). To determine whether the C-terminus of GABA(B2) might target GABA(B) to neurites, cultured hippocampal neurons were transfected with the truncated GABA(B2) constructs. Truncation of GABA(B2) at residue 841 resulted in primarily somatic localization; furthermore, axonal trafficking of this construct was significantly more restricted than dendritic trafficking. Finally, to biochemically assess trafficking of the truncated GABA(B2) constructs, we digested transfected HEK293 cell lysates with endoglycosidase H. When GABA(B2) was truncated at residue 841, it became sensitive to digestion by this enzyme, indicating incomplete trafficking. Taken together, these data show that the region of the GABA(B2) C-terminus between residues 841 and 862 is important for regulating forward trafficking and neuronal targeting of the GABA(B) receptor.

Drotrecogin alfa (activated) in severe falciparum malaria.

Drotrecogin alfa (activated) is a drug licensed for the treatment of severe sepsis. We describe the care of a 61-year-old man who developed multi-organ failure secondary to severe falciparum malaria infection with parasitaemia levels of 40%. Included in his care were an exchange blood transfusion and an infusion of Drotrecogin alfa (activated). Within hours of starting the infusion of Drotrecogin alfa (activated), the patient's clinical condition stopped deteriorating. Steady improvement followed with weaning from ventilatory assistance on day 14 post admission. The patient made a full recovery and was discharged home following rehabilitation. The indications for Drotrecogin alfa (activated) and the appropriateness of its use in severe malaria with multi-organ failure are discussed. Drotrecogin alfa (activated) may be a useful treatment in patients with multi-organ failure resulting from severe malaria.

Successful treatment of essential thrombocythaemia and recurrent abortion with alpha interferon.

A 27-year-old woman with essential thrombocythaemia (ET) had three miscarriages. She was treated with alpha interferon which was continued after she became pregnant again. The platelet count remained well controlled and at term she delivered a healthy baby with normal blood counts. The case demonstrates the effectiveness of IFN in preventing recurrent miscarriage associated with ET and the safety of IFN with respect to fertility and fetal development.

Use of photosensitive, antibody directed liposomes to destroy target populations of cells in bone marrow: a potential purging method for autologous bone marrow transplantation.

Liposomes containing the photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) were coupled to polyclonal sheep anti-mouse-Ig antibody and bound to cells coated with specific mouse monoclonal antibody. When illuminated with red light, the AlSPc in the liposomes was activated to produce singlet oxygen and the antibody and liposome targeted cells were destroyed. DW-BCL cells (an Epstein Barr virus immortalised B-cell line) were targeted with an anti-B-cell antibody (8A) and killed specifically, both alone and in the presence of bone marrow mononuclear cells (BM-cells), without phototoxic effects on the untargeted bone marrow CFU-GM progenitor cells. The presence of an excess of non-target cells did not interfere with antibody and liposome binding, or light access to target cells. Similar results were obtained with T-lymphocytes as target cells using anti-CD3 antibody. Specific targeting to the B-cells was demonstrated in the cell mixtures by use of fluorescent microscopy combined with a sensitive technique to detect low levels of AlSPc fluorescence, a cooled charge couple device (CCD) camera. This was also able to show low levels of non-specific background binding of AlSPc to BM-cells and a small population of cells that took up AlSPc in the absence of antibody. The latter were shown to be monocytes by flow cytometry.

Enhanced fluorescence in indirect immunophenotyping by the use of fluorescent liposomes.

Small unilamellar liposomes were optimised for cell phenotyping by indirect immunofluorescence. This involved selection and covalent attachment to the liposome of a polyspecific ligand for cell-bound antibody. For this purpose sheep anti-mouse antibody was preferred to protein A because of its ability to attach to cell-bound IgG1 as well as IgG2 at physiological pH. The maximally fluorescent concentration of encapsulated carboxyfluorescein was determined to be 20 mM and liposomes thus comprised gave up to a nine-fold increase in mean cell fluorescence when compared with sheep anti-mouse antibody conjugated to fluorescein isothiocyanate. There was no parallel increase in background fluorescence. Liposomes retained their targeting and fluorescence properties after 3 months storage. They could be sterilised and were as versatile in use as FITC-antibody conjugates.

Specific targeting and toxicity of sulphonated aluminium phthalocyanine photosensitised liposomes directed to cells by monoclonal antibody in vitro.

A partially purified fraction of the water soluble photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) was encapsulated in liposomes which were then linked to a targeting monoclonal antibody 791T/36 using a heterobifunctional linking agent. The photocytotoxic effects of the liposomes were determined on two cell lines bearing an antigen with which the targeting antibody binds: 791T, an osteosarcoma and C170, a colorectal carcinoma; and a control cell line not bearing the antigen; DW-BCL, an Epstein-Barr virus immortalised B-cell line. Antibody dependent cytotoxicity was observed in 791T and C170 cells and was proportional to the number of antigens on the cells, the AlSPc concentration and the time of exposure to activating red light. No significant toxicity was seen using untargeted liposomes, control cells or free AlSPc fraction under similar conditions. Targeted cells and controls kept in the dark also showed no significant toxicity. A possible mechanism of action is postulated and simple adaptations which demonstrate the versatility of the model are discussed. Some suggestions as to the clinical situations to which this system might be applied in the form of photodynamic therapy (PDT) are made.

Mixed function oxidase and UDP-glucuronyltransferase activities in the human Hep G2 hepatoma cell line.

In cultured human hepatoma cells phenolphthalein glucuronidation was increased 3-fold by 2 mM phenobarbitone (PB) in the culture medium but not by 25 microM benz(a)anthracene (BA), while 1-naphthol glucuronidation was not increased by either PB or BA. Ethoxyresorufin O-deethylation (EROD) was increased 15-fold by BA but not by PB, while the O-dealkylations of pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) were increased by either PB or BA. The BROD activity increased by BA was sensitive to inhibition by alpha-naphthoflavone whereas that induced by PB was not. This suggests induction of different cytochrome P-450 isoenzymes. Control Hep G2 cells had similar glucuronide conjugation and cytochrome reductase activities to freshly isolated human adult hepatocytes, but had lower O-dealkylation and elevated microsomal epoxide hydrolase activities.

Alpha-interferon therapy for essential thrombocythaemia.

18 patients with symptomatic essential thrombocythaemia were treated with recombinant alpha interferon (2a or 2b). Subcutaneous dosage regimens, which were well-tolerated, selectively lowered the platelet count and relieved symptoms in all patients, whether previously treated or untreated. Recombinant alpha-interferon may offer a non-leukaemogenic alternative therapy for these patients.

Uptake of antibody directed cytotoxic liposomes by CD3 on human T cells.

Using polyclonal human T cells and anti-CD3 monoclonal antibodies we have shown that small unilamellar liposomes covalently coupled with protein A become bound to T cells and not to B cells and that the binding was a specific liposome-antibody-receptor interaction. Intracellular delivery of liposome contents was demonstrated by the use of encapsulated carboxyfluorescein and flow cytometry and the transfer of membrane-bound liposomal carboxyfluorescein was virtually complete in 30 min. Liposomes containing methotrexate inhibited the growth of PHA-stimulated peripheral blood lymphocytes by 90%, after 48 h incubation. Potential applications are proposed in the study of the behaviour of surface membrane components and in T cell depletion and purging of bone marrow.

Coronary revascularization in the elderly patient.

A total of 1,275 elderly patients (70 years and older) underwent coronary artery bypass alone from 1970 to 1981. The percent of elderly patients who underwent coronary bypass surgery alone increased from 2.04% in 1971 to 8.2% in 1981. Most of the patients had severe, disabling or unstable angina pectoris. The overall early mortality rate was 5.8%. The early mortality rate was 13.9% in the first group (1970 to 1975) of 158 patients compared with 4.7% in the second group (1976 to 1981) of 1,117 patients. An average of 3.1 bypass grafts per patient were implanted. On follow-up examination, angina was relieved or decreased in 89% of the patients. The 5 year survival rate was 80.6% and the 10 year survival rate was 44.1%, with an average attrition of 3.9 and 5.6%/year, respectively. It is concluded that elderly patients are high risk surgical candidates, yet the risk has decreased progressively because of improved techniques of medical and surgical management and myocardial preservation. This decreasing operative mortality rate provides evidence that when medical management of the elderly patient with severe angina fails, coronary artery bypass becomes a successful alternative.

The morphology of ascending aortic aneurysms.

To determine the frequency of morphologic abnormalities of the aorta, especially of the media, in patients with aneurysms of the ascending aorta, tissue specimens from surgically resected ascending aortic aneurysms of 339 patients were studied. Included were 232 men and 107 women; 53 (29 men, 23 women) had clinical signs of Marfan's disease. Features evaluated and graded included fragmentation of elastic fibers, cystic medial change, medial fibrosis, medial necrosis, atherosclerosis, periaortic fibrosis, and thickening of the vasa vasorum. Both elastic fragmentation and cystic medial change were present in a high percentage of patients. Cystic medial change was inversely correlated with increasing age of patients, especially in the group of patients without clinical evidence of Marfan's syndrome. Marked changes of these types in many younger patients without Marfan's syndrome could reflect a "tissue insufficiency" in early life that causes the aortic wall to weaken and dilate. Medial necrosis, fibrosis, and atherosclerosis were directly correlated with age. Hemodynamic events are considered to initiate injury and repair within the aortic wall. Dissection was more frequently seen with medial abnormalities than with atherosclerosis.

Basophils, histamine and gastric acid secretin in chronic myeloproliferative disorders.

Fourteen patients with polycythaemia rubra vera (PRV) and four with chronic granulocytic leukaemia (CGL) were studied to examine the relationship between basophil counts, blood histamine, gastric acid secretion and dyspepsia or peptic ulceration. Basophil counts and blood histamine were markedly raised in untreated CGL but only marginally increased in other cases. Blood histamine and basophil counts were well correlated. Gastric acid secretion was normal in 10 out of 11 cases, and did not correlate with blood histamine values. The previously reported high incidence of dyspepsia and peptic ulceration is examined and its mechanisms are discussed.