A gene region encoding a segment of the major surface protein, HBsAg, of hepatitis B virus was analyzed from serum samples after orthotopic liver transplantation of three hepatitis B virus chronic carrier patients treated with a human anti-hepatitis B virus monoclonal antibody (SDZ OST 577). Each of these three patients became HBsAg negative after transplantation and therapy with the human anti-hepatitis B virus monoclonal antibody but returned to HBsAg positivity (first detected 143,251 and 252 days after the transplantation). Polymerase chain reaction DNA amplification was performed on DNA from serum samples showing low levels of recurrent HBsAg and reduced antigen reactivity with SDZ OST 577 antibody. Polymerase chain reaction DNA included a 230-bp highly conserved, major S gene region that was cloned into M13 bacteriophage; analysis of this DNA segment provided a consensus of DNA sequences for the serum samples exhibiting altered reactivity with the therapeutic monoclonal. Analysis of independent DNA clones from serum samples of patients exhibiting low but detectable recurrent serum levels of posttherapy HBsAg revealed the presence of S protein variant sequences when compared with polymerase chain reaction DNA derived from the original infected liver or pretherapy serum HBsAg. Genetic variation was predominant in a highly conserved peptide domain that has previously been implicated in antibody binding and neutralizing antibody epitopes. In independent patients infected with either adw or ayw hepatitis B virus subtypes, single nucleotide changes resulted in one to two amino acid differences for each variant allele (residues 124, 129, 131, 137, 140 and/or 145) when compared with pretherapy viral DNA. Administration of serum containing one of these variant viruses to a single hepatitis B-naive chimpanzee resulted in subclinical hepatitis and detectable levels of circulating anti-HBs and anti-HBc antibodies 49 and 70 days after virus administration, respectively. Hepatitis B virus DNA was recovered on liver biopsy between 6 and 8 wk after inoculation, although the animal remained persistently seronegative for HBsAg. DNA sequence analysis of both primate and patient liver hepatitis B virus confirmed the presence of the DNA encoding the S protein variant and associates this DNA with the predominant hepatotropic virus in liver infection.

Advanced glycoxidation end products (AGEs) are implicated in delayed diabetic wound healing. To test the role of diet-derived AGE on the rate of wound healing, we placed female db/db (؉/؉) (n ‫؍‬ 55, 12 weeks old) and age-matched control db/db (؉/؊) mice (n ‫؍‬ 45) on two diets that differed only in AGE content (high [H-AGE] versus low [L-AGE] ratio, 5:1) for 3 months. Full-thickness skin wounds (1 cm) were examined histologically and for wound closure. Serum 24-h urine and skin samples were monitored for N ⑀ -carboxymethyl-lysine and methylglyoxal derivatives by enzyme-linked immunosorbent assays. L-AGE-fed mice displayed more rapid wound closure at days 7 and 14 (P < 0.005) and were closed completely by day 21 compared with H-AGE nonhealed wounds. Serum AGE levels increased by 53% in H-AGE mice and decreased by 7.8% in L-AGE mice (P < 0.04) from baseline. L-AGE mice wounds exhibited lower skin AGE deposits, increased epithelialization, angiogenesis, inflammation, granulation tissue deposition, and enhanced collagen organization up to day 21, compared with H-AGE mice. Reepithelialization was the dominant mode of wound closure in H-AGE mice compared with wound contraction that prevailed in L-AGE mice. Thus, increased diet-derived AGE intake may be a significant retardant of wound closure in diabetic mice; dietary AGE restriction may improve impaired diabetic wound healing. Diabetes 52:2805-2813, 2003 W ound healing is impaired in diabetes and constitutes a major cause for increased morbidity and mortality in patients with diabetes (1). The majority of nonhealing wounds often lead to amputation. The percentage of amputees increases with age as do the direct costs of their care, rehabilitation, and lost productivity (2) The exact cellular and molecular mechanisms underlying the pathogenesis of this complication are not fully elucidated (3,4). However, a number of hyperglycemiadependent factors have been identified, including the progressive accumulation of advanced glycation end products (AGEs) (5).Two well characterized compounds, N ⑀ -carboxymethyllysine (CML) and methylglyoxal (MG), derivatives of glucoseprotein or glucose-lipid interactions, serve as markers for AGE in a wide range of disorders related to diabetes, renal failure, and aging (6 -8). According to recent observations, AGEs can be introduced in the body by exogenous sources such as diet and possibly in amounts that exceed those caused by hyperglycemia alone. A direct correlation is shown between the amount of AGEs consumed and that found in the circulation (9,10). In vitro data show that food-derived AGEs, which include CML and MG derivatives, can mimic the actions of endogenously formed AGEs and can induce intracellular oxidative stress and inflammatory cell activation, in a manner reversible by antioxidants or anti-AGE agents (11). Animal studies have revealed a significant contribution to the total AGE pool and related pro-oxidant or proinflammatory processes by dietary AGE intake, including tissue damage; this seems to be preventable by restric...

The closure of ungrafted sacrococcygeal pilonidal sinus excisional wounds was studied in 15 patients. Wound punch biopsies were taken on a regular basis, and histologic sections were made. To document changes, computer-assisted morphometric image analysis was employed. Initial average wound depth was 37.8 +/- 4.6 mm, and complete closure (0 wound depth) was reached by 68 days. Wound contraction contributed 88 percent to wound closure, whereas the deposition of scar only contributed 12 percent. Maximum cells density within granulation tissue was reached by day 18. Myofibroblasts, identified by alpha-smooth muscle actin immunostaining, first appeared on day 11. Unlike those observed in laboratory animals, myofibroblasts were a minor cell population of granulation tissue, never exceeding 10 percent of the cells. The pattern of collagen fiber organization was documented by polarized light microscopy of Sirius red-stained sections. Early granulation tissue collagen fibers demonstrated a fine greenish birefringence, whereas more mature granulation tissue collagen fibers were thicker, displaying orange-yellowish birefringence. Myofibroblasts were associated exclusively with thicker collagen fibers, whereas fibroblasts were associated with both fine and thick collagen fibers. It is proposed that human wound contraction involves a volume change whereby normal dermal and adipose tissues are pulled into the defect by forces generated within fibroblasts.

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A highly sensitive and specific two-site immunoradiometric assay (IRMA) for hCG has been developed and applied to the detection of the hormone in the urine of normal nonpregnant and pregnant individuals. The IRMA uses a solid phase coupled monoclonal antibody to the hCG beta-subunit for extraction of hormone from urine. The hCG extracted is then directly quantified by the binding of an affinity purified and radiolabeled rabbit antibody that reacts with the unique COOH-terminal peptide region of the hCG beta-subunit. The assay is capable of reliably and accurately measuring as little as 0.01 ng hCG/ml urine without interference from hLH. Assays of urine from normal men and nonpregnant women of reproductive age indicated that most individuals did not have detectable levels of hCG immunoreactivity, although a minority had minute amounts, with a mean value of approximately 0.01 ng hCG/mg creatinine. In contrast, all normal menopausal women studied had easily detectable levels of hCG immunoreactivity in their urine, with a mean value of 0.123 ng hCG/mg creatinine. A study of the excretion of hCG from three men injected with hormone for treatment of infertility indicated that after the first 24 h, hCG was cleared with a single exponential rate and was detectable to a level of 0.01 ng/ml. Application of the IRMA to measurements of hCG in the urine of two artificially inseminated patients indicated that the method was capable of detecting pregnancy as early as 9 days postovulation. The extreme sensitivity and specificity of the IRMA for urinary hCG in conjunction with the simplicity of assay performance and specimen collection should provide a substantial advantage over currently available methods for detection of early pregnancy and tumor monitoring.

Monoclonal antibodies were prepared against the a and f8 subunits of human chorionic gonadotropin (hCG). Although all were selected on the basis of their ability to bind the intact hormone, each also bound one of the two subunits but not both. Using a solid phase double antibody system to measure the relative binding to sites on the surface of hCG, we observed that four ofthe five antibodies bound to different sites on the molecule. This information was correlated with the ability of each antibody to inhibit the biological activity of hCG. Of the five antibodies tested for their ability to inhibit hCG-induced stimulation of rat testes steroidogenesis in vitro, two proved to be potent inhibitors, whereas the other three had almost no effect. This inhibition of steroidogenesis was highly correlated with the ability of the antibodies to inhibit hCG binding to testes homogenates. Thus, we have begun to derive a scheme that describes the relative binding positions of individual monoclonal antibodies and receptor on hCG. The purified monoclonal antibodies were iodinated and employed to evaluate which antigenic sites on hCG remained free in hCG-receptor complexes. The data indicated that portions of the g subunit in hCG-receptor complexes were buried (i.e., failed to bind radiolabeled antibody), whereas other portions remained exposed (i.e., they bound radiolabeled antibody). Those antibodies that interacted with portions of hCG that became inaccessible in the receptor complex also blocked the biological actions of hCG, whereas those that interacted with exposed sites had little or no effect on activity. Although we did not find antibodies to the a subunit that would bind to the hormone-receptor complex, we found that one of the two antibodies specific to a subunit epitopes blocked the actions of the hormone. Both antigenic determinants on the at subunit appeared to be lost after the hCG-receptor complex had formed. These studies suggest that each hCG subunit participates in the hormone-receptor complex and that portions of the ,B subunit project from the surface of the receptor.Human chorionic gonadotropin (hCG) is a glycoprotein hormone, composed of a and ,B subunits, which interacts with gonadal receptors to stimulate steroid synthesis (1). The isolated subunits exhibit less than 0.1% of the activity of the intact hormone and, within the limits of highest purity available, are thought to lack biological activity (1-3). As the subunits recombine, they reacquire biological activity (4), and spectral studies indicate that the conformation of the subunits is altered when they are recombined (5), suggesting that unique structural properties of the intact hormone may be needed for activity. Because neither subunit is active alone, the relative roles of the subunits in the actions of the hormone have been difficult to assess. Conceivably, only one of the subunits comes into direct contact with the receptor.Although the binding of hCG and related gonadotropins to Leydig cells has been studied at length (1), data con...

The protective efficacy of a human monoclonal antibody directed against the a determinant of hepatitis B virus (HBV) surface antigen was studied in a chimpanzee. A single high dose of 5 mg/kg (body weight) of monoclonal antibody SDZ OST 577 was intravenously administered to a chimpanzee, followed by intravenous challenge with 103-5 chimpanzee infectious doses of a wild-type HBV, the MS-2 strain (ayw subtype). The passively acquired antibody to HBV surface antigen could be detected for 40 weeks. Serum HBV DNA tested by a "nested" polymerase chain reaction assay was negative through the 36th week after virus challenge but became positive by the 38th week. The chimpanzee subsequently developed acute hepatitis B -1 year after challenge. The nucleotide sequence of the a determinant of the surface gene of the replicated virus was identical with that of the inoculated wild-type virus. Thus, a human monoclonal antibody directed against the a determinant ofHBV surface antigen delayed but did not prevent experimental infection of HBV and hepatitis in the chimpanzee. Our results indicate an incomplete ability of this antibody to protect against HBV infection in vivo after a single infusion.