Th1/Th2-like immunity and resistance to herpes simplex labialis.

To investigate potential immunologic mechanisms of resistance to recurrent herpes simplex labialis, we assayed serum antibody titers and cultured peripheral blood mononuclear cell (PBMC) cytokine production among patients with a history of frequent episodes (H+S+), herpes simplex virus (HSV)-seropositive individuals without a history of herpes labialis (H-S+) and HSV-seronegative persons (H-S-). In addition, H+S+ patients were exposed to experimental ultraviolet radiation (UVR) on the lips and the immunologic assay results compared among those who developed experimental lesions and those who did not. H+S+ patients were found to have higher median serum titers of HSV antibody and trends to lower levels of HSV-specific PBMC IFN-gamma and IL-2 than H-S+ control patients (123 vs 66, P = 0.04; 424 vs 548 pg/ml, P = 0.08; 14 vs 26 pg/ml, P = 0.14, respectively). Correlation of the results with the occurrence of experimental lesions showed the inverse: the subgroup of H+S+ patients with UVR-induced lesions had lower titers of antibody and trends to higher levels of IFN-gamma and IL-2 than H+S+ patients who could not be induced (93 vs 149, P = 0.02; 501 vs 347 pg/ml, P = NS; 26 vs 11 pg/ml, P = NS, respectively). The size and duration of UVR-induced lesions showed positive correlations with IFN-gamma and IL-2 levels (r = 0.60-0.67, P = 0.02-0.04). Although the small number of patients limited the power of this study, the overall pattern of the findings suggests that a Th1-like cytokine response (IFN-gamma and IL-2 production) may be associated with resistance to naturally occurring episodes of herpes labialis. The development and severity of experimental UVR-induced herpes labialis appears to be regulated differently and may involve an immunopathologic mechanism.

Baculovirus-expressed herpes simplex virus type 2 glycoprotein D is immunogenic and protective against lethal HSV challenge.

Herpes simplex virus type 2 glycoprotein D (gD2) was cloned and expressed in the baculovirus-Spodoptera frugiperda system. Milligram quantities of glycoprotein were recovered from suspension culture and subjected to purification by ion-exchange and immunoaffinity chromatography. The resultant purified gD existed as a homogeneous 57,500 MW monomeric species demonstrating reactivity with anti-gD monoclonal antibodies including those directed at a non-sequential neutralizing epitope of gD. Immunization of Balb/c mice with doses of 0.1-10.0 micrograms of AlPO4-absorbed gD resulted in elicitation of humoral and cellular responses to both HSV1 and HSV2 as well as to purified gD1 and gD2. Immunized mice receiving an infectious dose of 1 x 10(6) p.f.u. of HSV2 via the footpad route were significantly protected against infection at all doses tested when compared with unimmunized AlPO4 and uninoculated control animals.

Native herpes simplex virus glycoprotein D vaccine: immunogenicity and protection in animal models.

Mice, guinea pigs, and rhesus monkeys were immunized with immunoaffinity-purified native glycoprotein D (gD) derived from herpes simplex virus type 1 (HSV1). The native glycoprotein has evoked significant in vivo responses even at low doses. Thus, mice immunized with doses as low as 1 microgram were significantly protected from the morbidity and mortality of lethal HSV2 challenge and from establishment of latent HSV2 infection. Protection was dose-related and correlated with prechallenge serum neutralizing antibody titres to HSV. Similarly, immunized guinea-pigs demonstrated significant reductions in the frequency, severity and duration of genital lesions induced by HSV2 vaginal challenge. In long term immunogenicity studies, immunized rhesus monkeys exhibited significant serum neutralizing antibody responses to both HSV1 and HSV2. In vitro stimulation of monkey peripheral blood leucocytes with purified gD resulted in a significant cellular proliferative response. The results obtained in these animal models with a gD subunit vaccine provide an appropriate foundation for the initiation of human studies.

The bovine lens neutral proteinase comprises a family of cysteine-dependent proteolytic activities.

Inhibitor studies with peptide substrates demonstrate that bovine lens neutral proteinase comprises three distinct activities. Diisopropylfluorophosphate distinguishes the activity hydrolyzing carbobenzoxy-Gly-Gly-Leu-p-nitroanilide (inhibited) from that hydrolyzing carbobenzoxy-Leu-Leu-Glu-2-naphthylamide (not inhibited). Leupeptin inhibits hydrolysis of the substrate carbobenzoxy-Leu-Leu-Arg-2-naphthylamide, but not hydrolysis of carbobenzoxy-Gly-Gly-Leu-p-nitroanilide or carbobenzoxy-Leu-Leu-Glu-2-naphthylamide, demonstrating the presence of the third activity. Inhibition of the three activities by thiol reagents suggests that each activity may be dependent on an active-site cysteine residue.

Differential inhibition of two proteolytic activities in bovine lens neutral-proteinase preparations.

Hydrolysis of carbobenzoxy-Leu-Leu-Glu 2-naphthylamide by bovine lens neutral-proteinase preparations is not affected by the esterase inhibitor di-isopropyl fluorophosphate, whereas hydrolysis of carbobenzoxy-Gly-Gly-Leu p-nitroanilide is completely inhibited. Hydrolysis of alpha-crystallin, a lens structural protein, can be inhibited by only 50% after prolonged treatment with di-isopropyl fluorophosphate. These data suggest that the lens neutral-proteinase preparation contains at least two enzymes, one of which may be a serine proteinase. This may account, in part, for the previously observed complex response of the preparation to inhibitors.

Recovery of native proteins from preparative electrophoresis gel slices by reverse polarity elution.

A technique for high yield recovery of native, biologically active proteins from preparative polyacrylamide gel slices by reverse polarity elution is described. No apparatus other than the standard slab gel electrophoresis system is required. Several proteins have been recovered in biologically active form at a 90% yield, in quantities ranging from 0.4 mg to 4.2 mg. The method is effective with both small (9,000 dalton) and large (186,000 dalton) polypeptides. Both simple and complex proteins are recovered intact. For example, the copper-zinc and manganese superoxide dismutases from crude soybean extracts are active upon recovery. Similarly, the vitamin D-dependent calcium binding proteins from rat kidney and intestine are isolated by this method in homogeneous, active form.

Inhibition of superoxide production in human neutrophils by purified soybean polypeptides. Re-evaluation of the involvement of proteases.

Inhibition of neutrophil superoxide production has been previously reported for reagents and polypeptides which also inhibit serine proteases. There are disagreements between the results of different laboratories including our own, which have attempted to use the Kunitz soybean trypsin inhibitor to block neutrophil superoxide production. Having confirmed that crude extracts of soybean do contain inhibitory factors which affect neutrophil superoxide production, we have resolved polypeptides in an ethanolic extract of soybean flour by anion and cation exchange chromatography and preparative polyacrylamide gel electrophoresis. Fractions have been assayed for protease inhibitory activity and inhibition of neutrophil superoxide production. We have found an inverse relation between these two inhibitory activities during the purification process. Two of three isolated polypeptides are potent inhibitors of neutrophil superoxide production (50% inhibition at 10(-7) M) but retain only weak anti-trypsin activity. A third polypeptide is a potent inhibitor of trypsin but is completely lacking superoxide inhibitory activity. None of the isolated polypeptides inhibit chymotrypsin. The implications of these findings for the hypothetical association between neutrophil production of superoxide and cellular proteases are discussed.

Pseudo-inhibitors of neutrophil superoxide production: evidence that soybean-derived polypeptides are superoxide dismutases.

We have previously reported the purification of polypeptides from soybean which are potent inhibitors of superoxide production by human neutrophils. We now report that neither oxygen uptake nor hydrogen peroxide production by stimulated neutrophils is affected by these inhibitors. Furthermore, the E-1 and E-3 polypeptides inhibit ferricytochrome c reduction by a xanthine oxidase superoxide generation system. The inhibitory activity of E-3 in the model system is blocked by 1 mM KCN while E-1 is only slightly cyanide sensitive. Atomic absorption analysis of E-1 and E-3 polypeptides reveal copper in the latter and manganese in the former. Thus, E-3 is a copper-containing superoxide dismutase while E-1 appears to be a manganese-containing superoxide dismutase.

On the mechanism of membrane damage by C: exposure of hydrophobic sites on activated C proteins.

In previous papers we have presented evidence that peptides from C proteins C5b, C7, C8, and C9 become inserted in the lipid bilayer membranes and form a transmembrane channel. Presumably, this insertion follows exposure of hydrophobic domains by C activation. In the present experiments liposomes were made with 14C-phosphatidyl choline (PC) and Forssman antigen in the bilayer, and with 86Rb+ in the aqueous compartments. When such liposomes were incubated with anti-Forssman antibody (A) and guinea pig serum (GPS) as a source of C, substantially more 14C-PC and 86Rb+ were released than from liposomes treated with A and C4-deficient GPS, or with A and heated C, or with C alone, or with A alone. The specific release of PC was dependent on the dose of C. Prior treatment of GPS with cobra venom factor abolished its capacity to release PC. The release of PC by A and C7-deficient human serum (C7D-HS) was the same as that of GPS alone, i.e., there was no specific release. A and C8D-HS produced much less specific release than A and GPS; addition of purified guinea pig C7 or C8 to C7D-HS or C8D-HS, respectively, restored the PC release to its full extent. Hence, part of the PC removal is mediated by C5b,6,7; the remainder is attributable to C8 and/or C9.

On the mechanism of cell membrane damage by complement: evidence on insertion of polypeptide chains from C8 and C9 into the lipid bilayer of erythrocytes.

The preceding paper (Hammer, C.H., A. Nicholson, and M. M. Mayer, 1975, Proc. Natl. Acad. Sci., 72:5076) presented evidence on insertion of polypeptide chains from the C5b and C7 subunits of C5b, 6, 7 complex into the phospholipid bilayer of erythrocyte membranes. In the present study, EAC1-8 and EAC1-9 (sheep erythrocytes carrying rabbit antibody and complement proteins C1 through C8 or C9, respectively), prepared with either 125I-C8 or 125I-C9, were incubated with trypsin or chymotrypsin and the release of 125I was measured. Only 9 to 19% of the specifically bound radioactivity was released. In addition, elution experiments were performed with 0.02 M EDTA-1.0 M NaCl. This solution did not elute C9 from EAC1-9. By contrast cellbound C9 was recovered from erythrocyte membranes with sodium dodecyl sulfate (SDS). Thus, enzymatic stripping and elution experiments indicate that cellbound C9 behaves like an integral membrane protein, presumably due to insertion into the lipid bilayer. EAC1-9 membranes that had been subjected to extended digestion with trypsin or chymotrypsin were extracted with SDS to recover the enzyme-resistant part of the C9 molecule from the membrane. Even though this domain of C9 carried 90% of the radioiodine associated with native C9, its m.w. was found to be only 18,000 daltons by analysis on SDS-PAGE. This represents one-quarter of the native C9 molecule.

Interaction of phenols with old yellow enzyme. Physical evidence for charge-transfer complexes.

Evidence is presented that the changes in absorption spectrum obtained on complex formation between Old Yellow Enzyme and phenolic compounds are due to charge-transfer interactions. The positive correlation between the energy of the long wavelength transition and the Hammett para constant with a series of para-substituted phenols indicates that the phenol is the charge-transfer donor and the oxidized flavin of the enzyme is the charge-transfer acceptor. The same conclusion is drawn from studies in which the flavin of the native enzyme, flavin mononucleotide, was replaced by a variety of artificial flavins of different oxidation-reduction potential. The effect of pH on the dissociation constant for the enzyme-ligand binding also indicates that it is the phenolate anion, rather than the conjugate acid, which is responsible for the charge-transfer interaction. The significance of these results is discussed relative to long wavelength absorbing species detected with other flavoproteins.

Purification of intact old yellow enzyme using an affinity matrix for the sole chromatographic step.

Old Yellow Enzyme has been purified in high yield from crude extracts of brewers bottom yeast using an affinity matrix for the sole chromatographic step. The results of sodium dodecyl sulfate gel electrophoresis indicate that the enzyme is nearly homogeneous and consists of identically sized subunits of molecular weight of about 49,000. However, when the enzyme is isolated without the protective presence of a protease inhibitor, the electrophoretic pattern comprises three bands, which suggests limited, perhaps single bond cleavage of the polypeptide chain. This proteolytic cleavage results in weaker binding of ligands to the enzyme. The binding of a large number of compounds to the enzyme has been investigated, particularly with reference to the concomitant appearance of long wavelength absorption bands. The structural requirements for formation of long wavelength absorption are that the ligand must be aromatic in character, with an ionizable hydroxyl or thiol substituent.

A new activity of complement component C3: cell-bound C3b potentiates lysis of erythrocytes by C5b,6 and terminal components.

EAC4b,3b (sheep erythrocytes carrying rabbit antibody and guinea pig complement component fragments C4b and C3) adsorb human C5b,6 reversibly; the avidity of binding varies inversely with ionic strength. We believe that the receptor of C5b,6 is contributed by the cell-bound C3b because the binding capacity of EAC4b,3b varies with C3b multiplicity and can be blocked with rabbit antibody to guinea pig C3. The fixation of C5b,6 to the erythrocyte-bound C3b serves to concentrate C5b,6 on the cell surface; as a consequence, the hemolytic efficiency of C5b,6 is almost 100 times greater when assayed with EAC4b,3b than with plain erythrocytes. This potentiation represents a hitherto unrecognized function of cell-bound C3b.

Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement.

The ion permeability of planar lipid bilayers, as measured electrically, was found to increase modestly upon treatment with purified complement complex C5b,6 and complement components C7 and C8. The subsequent addition C9 greatly amplified this change. No permeability changes occurred when components were added individually to the membrane, or when they were used in paired combinations, or when C5b, C7, C8, and C9 were admixed prior to addition. Thus, there is a significant parallel between the permeability changes induced in the model membrane and damage produced in biological membranes by the C5b-9 complement attack sequence. The efficiency of membrane action by C5b-9 was critically dependent on the order in whcih components were added to the membrane. There were also differences in the electrical properties of membranes treated with C5b-8 and C5b-9, though in both cases the enhanced bilayer permeability is best attributed to the formation of trans-membrane channels. Collectively, the data are consistent with the hypothesis that the mechanism of membrane action by complement involves the production of a stable channel across the lipid bilayer, resulting in cell death by colloid-osmotic lysis.