Recombinant human acid alpha-glucosidase: high level production in mouse milk, biochemical characteristics, correction of enzyme deficiency in GSDII KO mice

Glycogen storage disease type II (GSDII) is caused by lysosomal acid alpha-glucosidase deficiency. Patients have a rapidly fatal or slowly progressive impairment of muscle function. Enzyme replacement therapy is under investigation. For large-scale, cost-effective production of recombinant human acid alpha-glucosidase in the milk of transgenic animals, we have fused the human acid alpha-glucosidase gene to 6.3 kb of the bovine alphaS1-casein gene promoter and have tested the performance of this transgene in mice. The highest production level reached was 2 mg/ml. The major fraction of the purified recombinant enzyme has a molecular mass of 110 kDa and resembles the natural acid alpha-glucosidase precursor from human urine and the recombinant precursor secreted by CHO cells, with respect to pH optimum, Km, Vmax, N-terminal amino acid sequence and glycosylation pattern. The therapeutic potential of the recombinant enzyme produced in milk is demonstrated in vitro and in vivo. The precursor is taken up in a mannose 6-phosphate receptor-dependent manner by cultured fibroblasts, is converted to mature enzyme of 76 kDa and depletes the glycogen deposit in fibroblasts of patients. When injected intravenously, the milk enzyme corrects the acid alpha-glucosidase deficiency in heart and skeletal muscle of GSDII knockout mice.     

Interaction of aldolase with pigeon erythrocyte plasma membranes]

Osmotically hemolysed pigeon erythrocytes retain a considerable part of the total cell content of aldolase activity. After washing off the ghosts from hemoglobin and removing the nuclei, a considerable portion of aldolase activity is found in the supernatant. The retained part of aldolase is rather firmly bound to plasma membranes (PM), as evidenced by the fact, that double washing with a mixture of 0.3 M sucrose, 0.01 M tris-HCl (pH 7.4) and 0.004 M MgCL2, or with 0.15 M NaCl or H2O does not appreciably decrease the aldolase activity of PM. Only washing of PM with 0.5 M NaCl results in appreciable decrease of aldolase retention by PM. The binding of aldolase proved to be temperature sensitive: after heating the binding of aldolase to PM specifically decreased. These data suggest that the interaction of the enzyme with PM of pigeon erythrocytes occurs in the intact cell and may be of physiological significance.     

Arthritis with an inflammatory dermatosis resembling Sweet's syndrome. Report of a unique case and review of the literature on arthritis associated with the inflammatory dermatoses

A patient with a unique case of chronic episodic arthritis coincident with flares of acneform, pustular, nodular and ulcerating skin lesions was observed over a five-year period. This patient and a review of the literature on arthritis associated with the inflammatory dermatoses provide evidence which may interrelate several of these nosologically confusing skin conditions, e.g., the family of leukocytoclastic angiitides with the newly posited acute febrile neutrophilic dermatosis of Sweet. Systemic manifestations and a variety of acneform, pustular, nodular and ulcerating cutaneous lesions in the inflammatory dermatoses are best explained by small vessel involvement, with individual syndromes being determined by the type and degree of vascular change. Perivascular neutrophilic infiltration is the unifying histologic feature of these small vessel diseases. Neutrophil infiltration differentiates these entities, and our patient, from the histologically nonspecific inflammations of the skin, e.g., Behcet's disease and pyoderma gangrenosum, which, although capable of causing identically appearing skin lesions, consist predominantly of lymphocytic dermal infiltrates even in the earlier stages. It appears important to recognized these morphologically varied acute inflammatory dermatoses with perivascular neutrophilic infiltration in view of their systemic features and the dramatic efficacy of corticosteroid therapy.     

A multiaperture collimator for use at 511 keV

The addition of autologous serum to mixtures containing human red cells, from pregnant and nonpregnant females, and sheep cells resulted in the formation of mixed aggregates containing both human and sheep red cells. In contrast, no aggregate formation occurred when autologous cord serum was added to mixtures containing cord red cells and sheep red cells. Heat inactivation of the adult serum or the presence of 0.15 M EDTA prevented the formation of mixed aggregates. These observations indicated that the mixed aggregates occurred through the complement-dependent red cell immune adherence (RCIA) phenomenon. The addition of untreated cord serum to mixtures containing inactivated adult serum restored the formation of mixed aggregates, indicating that the cord serum contained sufficient complement for RCIA. Natural antibody against sheep red cells was present in adult sera but was absent in cord sera. Using the RCIA receptor assay, the RCIA receptor activity of cord red cells was found to exceed significantly that of the adult pregnant cells (p less than 0.0025). It is postulated that this may represent an aspect of immune adaptation between mother and fetus.     

Symptomless hypoglycaemia

We report an unusual case of hypoglycaemia, in which a pregnant woman with a Rh-negative problem and random blood glucose levels of less than 10 mg/100 ml was found to have no clinical symptoms of hypoglycaemia. The patient, who had a blood glucose concentration of 7 mg/100 ml, behaved as normally as a person with the expected average physiological value. This finding did not seem to be connected in any way with the patient's serious Rh-ve problem. We have been unable to find reports of a similar situation in the literature available to us.     

Patterns of serial CEA assays and their clinical use in management of colorectal cancer

Through persistent clinical research efforts, the CEA test has developed into a useful although complex disease monitor for colorectal cancer. Although improved or prolonged survival from its use has not been demonstrated, CEA monitoring may allow more knowledgeable patient management. Several reports indicate that postoperative serial CEA assays may identify patients with early recurrence, especially when assays are done frequently. Patients with elevated pretreatment CEA levels usually showed progressively rising titers before other objective evidence of recurrence was apparent. A progressively rising CEA titer correlated well with recurrent cancer, but a normal CEA could not be used as proof of its absence. Persistently elevated CEA titers post-treatment was caused either by persistent disease or by nontumor-related factors. The CEA assay was not a substitute for clinical follow-up but was an adjunct in the diagnosis of eary recurrence. Patients with elevated CEA levels caused by localized disease treated by radiation therapy demonstrated a marked fall in serial CEA levels if all CEA-producing tumor was localized within the radiation portal. The use of pretreatment CEA values plus the pattern of CEA reponse to irradiation may help in the selection of fulguration versus abdominoperineal resection as primary treatment for rectal cancer. Persistently low serial CEA titers after irradiation therapy correlated with disease control. The use of frequent serial CEA assays in patients treated with chemotherapy compared well with other parameters as a monitor of disease progression or regression. When used with other clinical parameters, serial CEA trends appeared to be a useful adjunct in assessing the effectiveness of chemotherapy. A fall in circulating CEA or the stabilization of a rising titer after starting chemotherapy usually indicated an effective regimen, whereas a rising CEA titer may signal may signal the need to initiate or to change chemotherapy.     

Functional characterization of a stable, noncytolytic stage of macrophage activation in tumors

The state in which macrophages (Mphi) from regressing Moloney sarcomas could kill tumor target cells was a highly labile one which decayed rapidly in vitro. Thereafter, regressor Mphi were noncytolytic. Mphi from several different progressing sarcomas failed to kill, even when challenged with target cells immediately after explantation. Similarly, thioglycollate-induced peritoneal Mphi (TG-Mphi) did not kill. Noncytolygic Mphi derived either from progressing sarcomas or from long-term (up to 96 h) cultures of regressor Mphi were exquisitely sensitive to stimulation by bacterial lipopolysaccharide (LPS); picogram/milliliter amounts induced killing. Similar concentrations of LPS had no demonstrable effect on TG-Mphi. Thus, tumor Mphi generally appeared to have been primed in vivo, with those in regressing sarcomas having additionally acquired cytolytic activity. Inability of progressor Mphi to kill apparently stemmed from lack of, or failure to respond to, the signal needed in vivo to trigger cytolytic activity, rather than the total absence of activation.     

Detection of gluten in human sera by an enzyme immunoassay: comparison of dermatitis herpetiformis and celiac disease patients with normal controls

We have developed a triple sandwich enzyme immunoassay to detect circulating gluten in human sera. With human sera containing known amounts of added gluten as controls, the assay was sensitive in the range of 0.75 to 75 micrograms of gluten per ml of serum. Forty-one control subjects were compared to 21 patients with dermatitis herpetiformis and 11 patients with celiac disease. The dermatitis herpetiformis and celiac disease patients had significant elevation of serum gluten values over the control subjects. Circulating gluten antigenemia is a previously unrecognized feature which may be important in understanding the pathogenesis of dermatitis herpetiformis and celiac disease.