Antibody-dependent cell-mediated cytotoxicity in humans. I. Characterization of the effector cell.

Mononuclear cells from human peripheral blood are able to destroy alloantibody-coated target cells of a human lymphoblastoid line. The effector cell is a nonadherent, non-phagocytic, medium sized lymphoid cell. The cell is not detectable in early human fetuses. Its activity is destroyed by x-ray treatment in vitro but is not inhibited by anti-immunoglobulin sera. Its efficiency in antibody-dependent cell-mediated cytotoxicity is rapidly lost in culture and this loss is prevented by stimulation with allogeneic cells.     

Human ultralente insulin.

The greater solubility of human insulin and its possible faster action have led to doubts about whether a sufficiently long acting formulation could be produced to provide a basal supply for diabetics. In a double blind crossover study in 18 diabetics human ultralente insulin was as effective as beef ultralente insulin in controlling basal plasma glucose concentrations (median 5.7 mmol/l (103 mg/100 ml) with human and 6.3 mmol/l (114 mg/100 ml) with beef ultralente insulin respectively). There was no significant difference between human and bovine insulin in the rise in plasma glucose concentration from 0400 to 0700 after an injection the previous morning and no difference between patients receiving an adequate or insufficient dose of human ultralente insulin. Bovine insulin antibody binding was reduced with human insulin (p less than 0.002), which suggests that human insulin is less antigenic than beef insulin. Once daily human ultralente insulin provides a suitable formulation for the basal insulin requirement of diabetics.     

Human uracil-DNA glycosylase complements E. coli ung mutants.

We have previously isolated a cDNA encoding a human uracil-DNA glycosylase which is closely related to the bacterial and yeast enzymes. In vitro expression of this cDNA produced a protein with an apparent molecular weight of 34 K in agreement with the size predicted from the sequence data. The in vitro expressed protein exhibited uracil-DNA glycosylase activity. The close resemblance between the human and the bacterial enzyme raised the possibility that the human enzyme may be able to complement E. coli ung mutants. In order to test this hypothesis, the human uracil-DNA glycosylase cDNA was established in a bacterial expression vector. Expression of the human enzyme as a LacZ alpha-humUNG fusion protein was then studied in E. coli ung mutants. E. coli cells lacking uracil-DNA glycosylase activity exhibit a weak mutator phenotype and they are permissive for growth of phages with uracil-containing DNA. Here we show that the expression of human uracil-DNA glycosylase in E. coli can restore the wild type phenotype of ung mutants. These results demonstrate that the evolutionary conservation of the uracil-DNA glycosylase structure is also reflected in the conservation of the mechanism for removal of uracil from DNA.     

The human antimurine xenogeneic cytotoxic response. I. Dependence on responder antigen-presenting cells

We have examined the role of the human responder APC in the generation of CTL responses to xenogeneic antigens. Of six xenogeneic responses evaluated, only the human antimurine response was dependent on human APC for CTL generation. APC requirements for the other five xenogeneic responses more closely resembled those observed in the generation of human or murine alloreactive CTL. Depletion studies identified a defective human CD4+ Th cell-murine stimulator cell interaction that could be bypassed by the addition of exogenous IL-2. The function of the responder APC involved in the human antimurine CTL response was inhibited by chloroquine, suggesting a requirement for Ag processing. Effective presentation of murine stimulator Ag by human APC was completely blocked by anti-human Ia mAb, indicating that the Ag is presented to Th cells via the human class II molecule. These results are consistent with an Ia-dependent recognition of processed murine Ag by human T cells and represents a model for investigating human T cell activation requirements, Th cell function, and MHC restriction.     

Studies on polypeptides. V. Improved synthesis of human proinsulin C-peptide and its benzyloxycarbonyl derivative. Circular dichroism and immunological studies of human C-peptide.

An improved synthesis of human C-peptide is described. Five fragments: 33-39, 40-46, 47-49, 50-54 and 55-63 were used in the total synthesis. In the fully protected C-peptide the N-terminal alpha-amino function was blocked by a benzyloxycarbonyl group and the carboxyl and serine hydroxyl functions were blocked by t-butyl protection. The latter protecting groups were removed by trifluoroacetic acid to obtain N-alpha-benzyloxycarbonyl human C-peptide which, on catalytic hydrogenation, yielded human C-peptide. The immunoreactivity of the prepared human C-peptide was tested and found to deviate slightly from the human C-peptide synthesized earlier by another route. When tested in the immunoassay, human pancreatic extracts containing natural C-peptide (or fragments thereof) showed dilution patterns identical to that of the new synthetic C-peptide but different from that of the previously synthesized batch of C-peptide. The possible explantation for the observed differences in the immunoreactivity is discussed.     

Synergistic cytotoxicity. II. In vitro arming of monocytes and T cells by a heat labile fraction of human plasma.

In a previous paper we demonstrated that freshly obtained human plasma contain a heat labile nonantibody factor that induced human mononuclear cells to become nonspecifically cytotoxic toward xenogeneic but not allogeneic RBC targets. We now present evidence that this factor has a loose affinity for human monocytes and human T cells and can arm then to kill xenogeneic RBC targets. Furthermore, proteolytic enzymes markedly enhance this arming effect. This ability to be armed by a heat labile component found in fresh human plasma and the fact that proteolytic enzymes markedly enhance cytotoxicity clearly dissociate this model of nonspecific cytotoxicity for previously reported NK models.     

Human prothrombin activation.

Human prothrombin has been purified from American Red Cross Factor IX concentrates. Studies of the activation of the human prothrombin with the use of sodium dodecyl sulfate electrophoretic analysis of activation products indicated that human prothrombin activation is similar to bovine prothrombin activation. Molecular weight analysis of human prothrombin and intermediated by sodium dodecyl sulfate co-electrophoresis with bovine prothrombin and its intermediates resulted in molecular weights of 70,000 for prothrombin, 51,000 for intermediate 1, 41,000 for intermediate 2, 23,000 for intermediate 3, and 13,000 for intermediate 4. Amino acid compositions of human prothrombin and intermediates are similar to those for bovine prothrombin and intermediates. NH2-terminal sequence studies of human prothrombin, intermediates, and alpha-thrombin A and B chains placed the intermediates in the parent human prothrombin molecule as described for the bovine system. Intermediate 3 is the NH2-terminal of prothrombin, and intermediate 1 is the COOH-terminal segment of the zymogen. Intermediate 4 is the NH2-terminal of intermediate 1. Intermediate 2', the immediate precursor of alpha-thrombin, is the COOH-terminal segment of intermediate 1. In general, a high degree of homology in the primary structure of prothrombin and intermediates was observed between the human and bovine system. The NH2-terminal sequences of human intermediate 2' and alpha-thrombin A chain are identical. However, human intermediate 2' isolated in a manner identical with that used for the isolation of bovine intermediate 2 is homologous with bovine intermediate 2, beginning with residue 14.     

Studies on human kininogens. I. Isolation, characterization, and cleavage by plasma kallikrein of high molecular weight (HMW)-kininogen.

1. Human high molecular weight (HMW)-kininogen was highly purified from human plasma by chromatographies on QAE-Sephadex A-50 and CM-Sephadex C-50. Human HMW-kininogen thus purified was a mixture of a single chain and a disulfide-linked pair of chains. Human HMW-kininogen is an acidic glycoprotein having a molecular weight of 120,000. The amino acid composition of human HMW-kininogen is quite similar to that of bovine HMW-kininogen. 2. We investigated whether the liberation of kinin from human HMW-kininogen by human plasma kallikrein was accompanied by liberation of histidine-rich fragments, as observed with bovine HMW-kininogen (Han et al. (1975) J. Biochem. 77, 55--68). After prolonged incubation of human HMW-kininogen and human plasma kallikrein followed by gel-filtration on Sephadex G-50, a fragment of molecular weight 8,000 was isolated together with bradykinin. However, the histidine content of the fragment was not as high as that in the bovine fragments. Most of the histidine in human HMW-kininogen was recovered in the kinin-free protein, and the light chain of kinin-free protein was found to be rich in histidine compared with the heavy chain. These results suggest that the histidine-rich sequence in human HMW-kininogen is not released by the action of human plasma kallikrein, but remains bound to the light chain of kinin-free protein.     

Cellular immune responses in Huntington's disease (H.D.). Detection of H.D. and multiple sclerosis (M.S.) brain antigenicity by H.D. but not M.S. lymphocytes.

Lymphocytes from patients with Huntington's disease (H.D.) exhibit a cellular immune response which consists of two components: a minor component directed against antigen common to all human brains, and a major component directed against antigen found thus far only in H.D. and multiple sclerosis (M.S.) gray matter. M.S. lymphocytes exhibit a response to antigen common to all human brains, but fail to show any H.D. or M.S. brain-specific response.     

Isoform purification of gastric lipases. Towards crystallization.

Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.     

Isoforms of human cytochrome-c oxidase. Subunit composition and steady-state kinetic properties.

The subunit pattern and the steady-state kinetics of cytochrome-c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome-c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus-encoded subunits of the various cytochrome-c oxidase preparations. Tissue homogenates, in which cytochrome-c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome-c oxidase steady-state kinetics. Cytochrome-c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady-state reaction between human ferrocytochrome c and the four human cytochrome-c oxidase preparations revealed large differences for the low-affinity TNmax (maximal turnover number) value, ranging from 77 s-1 for kidney to 273 s-1 for liver cytochrome-c oxidase at pH 7.4, I = 18 mM. It is proposed that the low-affinity kinetic phase reflects an internal electron-transfer step. For the steady-state reaction of human heart cytochrome-c oxidase with human cytochrome c, Km and TNmax values of 9 microM and 114 s-1 were found, respectively, at high ionic strength (I = 200 mM, pH 7.4). Only minor differences were observed in the steady-state activity of the various human cytochrome-c oxidases. The interaction between human cytochrome-c oxidase and human cytochrome-c proved to be highly specific. At high ionic strength, a large decrease in steady-state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady-state TNmax and Km parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome-c oxidase deficiency.     

Cryptosporidium canis n. sp. from domestic dogs.

Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts approximately 90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.     

Genetics of somatic cell surface antigens. III. Further analysis of the AL marker.

The AL antigen present on the surface of various human somatic cells, and on those hybrids of human and Chinese hamster ovary cells which have retained human chromosome number 11, has been resolved into at least two separate antigenic activities, a1 and a2. Specific antisera active against each antigen separately have been prepared. By treatment of the original AL+ hybrid with mutagenic agents and selection in particular antisera, stable clones are preparable whose phenotypic behavior corresponds to the antigenic compositions a1+ a2-, a1- a2+, and a1- a2-. The adsorption behavior of these variants for specific antisera is consistent with their phenotypic assignments.     

Cell surface antigens. IV. Immunological coorespondence between glycophorin and the a1 human cell surface antigen.

A stable human-Chinese hamster ovary cell hybrid has been produced which, in addition to the complement of Chinese hamster ovary (CHO-K1) chromosomes, contains only one human chromosome, No. 11. The human cell-surface antigens whose expression is controlled by human chromosome 11, and are expressed by this hybrid, have been defined as the AL immunogenetic complex. Although one component of this immunogenetic complex (a1) is also expressed by human red blood cells, a second component (a2) is not. Killing of an a1+ hybrid by anti-a1 serum and complement can be completely inhibited by glycophorin, the major glycoprotein component of the human erythrocyte membrane. In the presence of complement, antiserum prepared against glycophorin will kill only those cells which express a1. The anti-a1 killing activity of the anti-glycophorin can be absorbed out only by those cells which express a1. Therefore, it is concluded that the a1 cell-surface antigen has at least one antigenic component in common with glycophorin.     

Immunosuppression by human gangliosides. II. Carbohydrate structure and inhibition of human NK activity.

Gangliosides shed by tumors enhance tumor formation, possibly by suppressing host antitumor immune function, and gangliosides purified from animal tissues and cultured cells inhibit human cellular immune function in vitro. Determination of immunosuppressive activity of highly purified gangliosides, to uncover structure-activity relationships, is therefore important. Here we have studied a series of gangliosides obtained from human tissue and determined their effects on human natural killer (NK) activity. Total gangliosides from human brain tissue were moderately inhibitory; 100 nmol/ml reduced NK activity of human nonadherent PBMC by 43%. The influence of carbohydrate structure upon inhibitory activity was determined by study of eight highly (HPLC) purified individual gangliosides. Of these, we unexpectedly found that the two minor brain gangliosides with the simplest carbohydrate structures, GM2 and GM3, were very active inhibitors (75 and 47%, respectively, at 50 nmol/ml). In contrast, the structurally more complex major species, GM1, GD1a, GD1b, GT1b, and two other minor gangliosides, GD2 and GD3, were inactive. Reduced effector-target binding in a single-cell binding assay by GM2 but not GM3 suggests different mechanisms of inhibition by these two active gangliosides. Since GM2 and GM3 are present in high concentrations in, and are shed by, several common human tumors (e.g., neuroblastoma, melanoma, and glioma), their ability to inhibit NK cytotoxicity supports the hypothesis of a role of shed tumor gangliosides in the enhancement of tumor formation.     

Relationship between enhanced reactivation and mutagenesis of u.v.-irradiated human cytomegalovirus in normal human cells.

The survival of u.v.-irradiated human cytomegalovirus (HCMV) on u.v.-irradiated human IAFP-1 cells was increased over that on unirradiated cells. Irradiated virus had a higher forward mutation frequency towards temperature sensitivity in irradiated than in unirradiated cells. Enhanced reactivation of u.v.-irradiated HCMV is thus mutagenic in normal human cells. This observation supports the possible induction of an error-prone mode of DNA repair in u.v.-irradiated mammalian cells.     

Construction and functional analysis of hybrid interleukin-6 variants. Characterization of the role of the C-terminus for species specificity.

We have constructed several hybrid human interleukin-6 (IL-6) variants in which the carboxyl-terminus, which includes a receptor binding site of IL-6 has been replaced with the C-terminus of various proteins homologous to human IL-6. IL-6 hybrids with the C-terminus of human growth hormone and human granulocyte-colony stimulating factor maintain part of the biological activity of human IL-6. Replacing the C-terminus of human IL-6 with the C-terminus of mouse and rat IL-6 resulted in a normal or increased activity on a mouse cell line; however, this gave a low (to 200-fold less) activity on a human cell line compared to wild-type human IL-6. We therefore conclude that the C-terminus of IL-6 plays an important role in the species specificity of IL-6.     

Adeno-associated virus vectors.

Adeno-associated virus is a human parvovirus that integrates its DNA genome into host cell chromosomes with very high efficiency. This suggests that adeno-associated virus may be a useful vector for human gene therapy. Interest in adeno-associated virus vectors increased greatly in the last year following reports that adeno-associated virus genome integration may be site specific and occur at preferred sites in the human genome. Several genes relevant to the treatment of genetic or infectious diseases have been expressed in adeno-associated virus vectors in vitro.     

Geometry of the human erythrocyte. I. Effect of albumin on cell geometry.

The effects of albumin on the geometry of human erythrocytes have been studied. Individual red cells, hanging on edge from coverslips were photographed. Enlarged cell profiles were digitized using a Gradicon digitizer (Instronics Ltd., Stittsville, Ontario). Geometric parameters including diameter, area, volume, minimum cylindrical diameter, sphericity index, swelling index, maximum and minimum cell thickness, were calculated for each cell using a CDC 6400 computer. Maximum effect of human serum albumin was reached at about 1 g/liter. Studies of cell populations showed decreases in mean cell diameter of up to 6%, area 6%, and volume 15%, varying from sample to sample. The thickness of the rim was increased while that at the dimple was decreased. Studies of single cells showed that area and volume changes do not occur equally in all cells. Cells with lower sphericity indices showed larger effects. In the presence of albumin, up to 50% of the cells assumed cup-shapes (stomatocytes). These cells had smaller volumes but the same area as biconcave cells. Mechanical agitation could reversibly induce biconcave cells to assume cup shapes without area or volume changes. Experiments with de-fatted human albumins showed that the presence of bound fatty acids in varying concentrations does not alter the observed effects. Bovine serum albumin has similar effects on human erythrocytes as human serum albumin.