Carlos George-Nascimento

Antigenicity and immunogenicity of domains of the human immunodeficiency virus (HIV) envelope polypeptide expressed in the yeast Saccharomyces cerevisiae.

Expression vectors were constructed for the production of various domains of the envelope gene product of the SF-2 isolate of human immunodeficiency virus (HIV) in the yeast Saccharomyces cerevisiae. Serum specimens from HIV seropositive blood donors reacted in immunoblot assays with recombinant polypeptides from both the gp120 and gp41 coding regions of env. Polypeptides from both domains were purified and injected into experimental animals. Antibodies raised in rabbits to env-2, a recombinant polypeptide representing the majority of the protein moiety of gp120, reacted with fully glycosylated native gp120 of HIV-SF2 virions. In addition, these env-2 antisera showed reactivity with viral gp120 of divergent HIV isolates. A 121 amino acid polypeptide (env-5), representing the region of gp41 stretching between the two hydrophobic domains of the protein, elicited antibodies in rabbits that reacted with glycosylated, native gp41. Thus, selected domains of the HIV env gene expressed in genetically engineered yeast, are recognized by sera from HIV infected humans, elicit antibodies that react with native HIV glycoproteins and provide a source of envelope antigens for evaluation as potential subunit vaccines for HIV.

Luminal hydrolysis of recombinant human epidermal growth factor in the rat gastrointestinal tract: Segmental and developmental differences

Epidermal growth factor (EGF), present in high concentrations in the milk of various species, is biologically active following oral administration to young animals. Although in vivo studies show gastrointestinal processing of dietary EGF during early postnatal development, the relative importance of luminal and mucosal digestion in such processing is undefined. To characterize the luminal metabolism of dietary EGF in the developing gastrointestinal tract, we incubated human recombinant 125I-EGF in vitro at 37 degrees with luminal fluid from the stomach and various segments of the small intestine of 12 day old suckling and 31 day old weanling rats and analyzed the resulting reaction products. The rate of EGF hydrolysis as determined by generation of acid soluble material was greater in weanling small intestine than in suckling, with maximal hydrolytic capacity observed in the mid-jejunum and ileum. Minimal hydrolysis was observed with stomach fluid from both age groups, and EGF retained its ability to elute as a single species on Sephadex G-25 columns and to bind to monospecific affinity columns and placental membrane receptors. Incubation with suckling small intestinal fluid produced little change in the chromatographic profile on Sephadex G-25, but a reduction in antibody and receptor binding was observed. In contrast, incubation with weanling small intestinal fluid yielded both a more pronounced loss of EGF-like material on G-25 columns and a greater reduction in receptor and antibody binding. We conclude that little luminal EGF degradation occurs in the rat stomach during the suckling and weanling periods, but that in the lumen of the small intestine breakdown increases during postnatal development.

Is Dietary Epidermal Growth Factor Absorbed by Premature Human Infants

Urinary epidermal growth factor (EGF) levels from infants receiving breast milk, a rich source of the growth factor, were compared with the levels excreted by infants receiving bovine milk based formulae or total parenteral nutrition that contains very little EGF. Although at 5-10 days after birth there was no significant difference in the urinary EGF output by the three groups of infants, by 13-17 days the urinary EGF output by breastfed infants was higher than by infants fed the two EGF-poor diets. These latter results are consistent with the hypothesis that EGF crosses the gastrointestinal wall to enter the general circulation in the suckling infant.

Subcutaneous but not Intraluminal Epidermal Growth Factor Stimulates Colonic Growth in Normal Adult Rats

Epidermal growth factor (EGF) was administered by chronic subcutaneous or intracolonic infusion into normal adult rats to determine the effect on colonic growth. Subcutaneous infusion of 200 micrograms EGF/kg/day for 7 days increased the cross-sectional mass and protein content of the muscularis and mucosal layers of the proximal colon, with the distal colon showing less response. In the mucosa, subcutaneous EGF induced proportional increments in the number of cells per crypt, and in the number of cells positively labelled for PCNA, while maintaining a normal crypt growth fraction. In contrast, an 8-fold higher dose of EGF administered intraluminally had no effect on colonic mucosal or muscularis growth. This lack of bioactivity was unlikely to reflect rapid luminal degradation as radiolabelled EGF remained stable in the colonic lumen for at least 4 h. The results demonstrate that the normal adult colon is responsive to subcutaneously delivered EGF, particularly the proximal colon, whereas EGF may not be active on the normal colon when presented from the luminal direction.

Hepatitis B virus e antigen specific epitopes and limitations of commercial anti-HBe immunoassays.

Current commercial hepatitis B virus (HBV) anti‐HBe immunoassays are designed so that anti‐HBe is detectable only in the absence of excess HBeAg. Recently, with the use of direct anti‐HBe assays, anti‐HBe was detected in individuals who had been seropositive for several years for HBeAg [Maruyama et al. (1993) J. Clin. Invest. 91:2586–2595]. Although anti‐HBe seroconversion does not necessarily indicate subsequent HBeAg clearance, the ability to detect earlier anti‐HBe seroconversion could have clinical significance for monitoring patients undergoing HBV immunotherapy (e.g., α interferon therapy). Because the HBeAg and the HBcAg share 149 amino acids, an anti‐HBe assay must distinguish anti‐HBe from anti‐HBc antibodies. Although the HBV HBeAg and HBcAg display distinct immunogenic determinants, much remains unknown regarding the complete epitope spectrum specific to each antigen. The goal of this study was 3‐fold. The first objective was to identify HBeAg specific linear epitopes. The second objective was to design an anti‐HBe immunoassay capable of detecting anti‐HBe specific antibody in the presence of excess HBeAg. The third objective was to characterize early anti‐HBe seroconversion antibodies. The major linear epitope residing in the HBeAg amino acid sequence was mapped and 2 novel minor epitopes (δ, γ) which appear to be HBeAg specific have been identified. An anti‐HBe immunoassay capable of detecting anti‐HBe specific antibody in the presence of excess HBeAg was designed. Finally, it was found that early anti‐HBe seroconversion antibodies appear to be conformational, whereas later seroconversion, more typically associated with the clearance of HBeAg, is characterized by the presence of antibodies to the linear HBeAg epitopes. J. Med. Virol. 60:256–263, 2000.

Intestinal Absorption of Epidermal Growth Factor in Newborn Lambs

Milk contains high concentrations of numerous growth factors including epidermal growth factor (EGF)1 and insulin-like growth factor-1 (IGF-1)2,3,4, observations that have led to the suggestion that milk growth factors may play an important role in infant growth and development.1,4 This would be possible only if growth factors survive digestion in the neonatal gastrointestinal tract. Moreover, direct actions on tissues other than the gut mucosa would further require that substantial amounts of milk growth factors are absorbed intact into the neonatal circulation.

Effects of Epidermal Growth Factor Administration on Repair of Acetic Acid-Induced Colonic Ulcerations in Rats

The effect of subcutaneous and luminal epidermal growth factor (EGF) administration on acetic acid-induced colonic ulceration was determined in adult rats. Application of acetic acid to the distal colonic lumen caused epithelial denudation, mucosal ulceration and inflammation in the exposed segment. Re-epithelialization was detectable 5 to 7 days later, with near-complete resolution of the lesion by 14 days post-injury. Luminal EGF (1.6 mg/kg bw/day) or subcutaneous EGF (200 micromilligrams/kg bw/day), administered for 4 or 6 days from the time of ulceration failed to enhance re-epithelialization of the acid-exposed segment. However, mucosal and submucosal thickening was attenuated 20-40% by subcutaneous EGF, reflecting a reduction in edema. Luminal EGF had a similar but less substantial effect in the submucosa, but was more effective at attenuating muscularis thickening adjacent to the lesion. In conclusion, administration of exogenous EGF for up to 6 days failed to enhance re-epithelialization of acetic acid-induced colonic ulcerations but did attenuate the associated edematous response.

Incomplete process of recombinant human platelet-derived growth factor produced in yeast and its effect on the biological activity.

Partially purified recombinant human Platelet-derived Growth Factor BB homodimer isolated from yeast culture media contains variable amounts of unprocessed PDGF-BB. This unprocessed PDGF-BB is found as a result of incomplete cleavage of the precursor to form the mature protein. Although the signal peptide is efficiently removed, a fraction of the PDGF secreted has an extended sequence corresponding to the truncated yeast alpha-factor leader. The data suggest that it is the amino acid chain from the truncated a-factor leader and not the sugar moiety attached to it that is responsible for the higher mitogenic activity found in this unprocessed molecule compared to highly purified PDGF-BB.