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Hepatitis e Antigen and Neonatal Immunologic Tolerance to Hepatitis B
May 1991
SELECTED SUMMARIES
and> 65-year age groups. The largest number of patients (31%) were seen by general and family practitioners, followed in declining order by internists (20%) and pediatricians (15%). Only 4% of all these patients were seen by gastroenterologists. The impact of constipation on health care costs and morbidity are considerable. Fortunately, constipation results in fewer hospitalizations and deaths than other digestive disorders-25.5 million hospital days per year for peptic ulcers and gallbladder disease combined compared with 13.6 million days per year for constipation. However, in the United States alone, cathartics are prescribed for more than three million people yearly, with an estimated cost in the millions of dollars, and more than 200 million dollars are spent annually for over-the-counter laxatives UClin Gastroenterol1989; 11:525-536). The complications of constipation are particularly a problem for the elderly and bedridden population. Potential morbid problems include fecal impaction, fecal incontinence, stercoral ulcerations, megacolon with volvulus, ischemia or perforation, rectal prolapse, hemorrhoids, and rarely cardiac and cerebrovascular dysfunction (N Engl JMed 1989;321:658-661). In most older patients, constipation can be effectively treated by increasing dietary fiber and fluids or by adding a bulk-forming agent such as psyllium. However, an important subgroup requires additional laxative therapy which must be safe and suitable for long-term use. More and more the most commonly prescribed drugs for these patients are the osmotic agents particularly the nonabsorbable sugars such as lactulose and sorbitol. Those sugars have an osmotic laxative effect probably related to their conversion to organic acids by colonic bacteria and the resulting reduction in stool pH (Gut 1972;13:859-866). Their only side effects are bloating, flatulence, and diarrhea. Lactulose has been shown in several studies to be safe and effective for treating constipation in the elderly (Gut 1968:84-86; JAm Geriatr Soc 1978;26:236-239; J Clin Gastroenterol 1981;3(Suppl 1):23-28). This study is the first report showing the effectiveness and safety of sorbitol in a similar group of constipated patients. Unfortunately, the practical value oflactulose for treating constipation in the elderly has been limited by its high cost. The cost to the pharmacist for a 960-mL bottle of lactulose ranges from $30 to $50. In contrast, a similar quantity of sorbitol costs approximately $5. Sorbitol is approved by the Food and Drug Administration as a safe and effective laxative. However, it is usually considered only for specialized uses such as counteracting the constipating effects of potassium-binding resins rather than for general use in constipation. Why should this be? The authors of this article correctly suggest that the major determinants for prescribing practices are commercial marketing and the scientific literature (Am J Med 1982;73:4-8). Therefore, inexpensive generic drugs such as sorbitol would not be expected to appear in the advertising pages of various medical journals. Furthermore, the shortage of noncommercial funding for clinical research reduces the likelihood of generic agents appearing in the scientific literature (Lancet 1987;1:257258). If effective "Brand X" drugs are not to be overlooked, health care organizations and private foundations concerned with the rising cost of medical care and medications may need to become involved in supporting research into promising generic drugs. The authors of this article are to be thanked for reawakening our awareness to an inexpensive alternative to lactulose in the treatment of constipation in the elderly. Sorbitol may also be effective therapy for women, younger patients, or those who are bedridden but these studies need to be performed. Nevertheless, the use of sorbitol should save money for elderly patients and their provider organizations. Because the use of lactulose has been restricted as a cost control measure by many health care providers, sorbitol may also improve patient access to optimal therapy.
J. E. RICHTER, M.D.
1475
Editor's Note. Although this study was randomized and double blind, it was not placebo controlled. Moreover, baseline stool frequency (prior to laxatives) was not measured. Therefore, the efficacy of sorbitol and lactulose as a laxative in these ambulatory, elderly men cannot be determined from this study.
HEPATITIS e ANTIGEN AND NEONATAL IMMUNOLOGIC TOLERANCE TO HEPATITIS B Milich DR, Jones JE, Hughes JL, et a1. (Departments of Molecular Biology, Immunology, and Molecular and Experimental Medicine, Scripps Clinic and Research Foundation, La Jolla, California). Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero? Proc Natl Acad Sci USA 1990;87:6599-6603. To examine the tolerogenic potential of hepatitis B virus (HBV) antigens, the authors produced hepatitis B e antigen (HBeAg)-expressing transgenic mice and used a neonatal tolerance system to investigate the tolerogenic characteristics of the hepatitis B core antigen (HBcAg) and the HBeAg. The authors produced an HBeAg-expressing transgenic mouse lineage designated B10.S Tg-31e that contained a HBV DNA construct (subtype ayw, coordinates 1804-2804) with the complete pre core plus core open-reading frame linked to the mouse metallothionein I promoter. HBeAg was present at a level of 8-10 nglmL in the serum of these mice, but the sera were unreactive in an HBcAg-specific enzymelinked immunosorbent assy (ELISA). T cells from these B10.S Tg-31 transgenic mice were unresponsive to HBeAg, HBcAg, and the immunodominant peptide p120-131 common to both antigens. Furthermore, these transgenic mice did not produce anti-HBe spontaneously. When immunized with a particulate form of HBeAg expressing both HBe and HBc antigenicity, the B10.S Tg-31e mice produced a primary and secondary antibody response to HBcAg but only a very weak secondary antibody response to HBeAg. In vitro studies demonstrated that these transgenic mice produced a normal immunoglobulin M (lgM) response to HBcAg but a greatly reduced IgG response to this antigen, suggesting a defect in T -helper function for anti-HBc B-cell responses. A single neonatal injection with HBcAg was found to induce transient T-cell tolerance to HBcAg and the common HBc/HBe peptide p120-131. Additional studies were performed to assess the T-cell responder status of nontransgenic offspring of B10.S Tg-31e transgenic mice. Pregnant HBeAg-expressing transgenic mice were administered zinc in a dose that increased serum HBeAg levels to approximately 72 ngimL. T-cell proliferative responses of transgenic and non transgenic offspring were assessed at 6 weeks of age and compared with those of 6-week-old control mice. As expected, the transgenic offspring demonstrated significant tolerance to HBeAg and peptide fragments of HBeAg. Whereas nontransgenic littermates were not completely tolerant to these antigens, T-cell proliferative responses of these mice to HBeAg and the p120-131 peptide fragment common to HBeAg and HBcAg were intermediate to those seen in control and transgenic mice of the same age.
1476
GASTROENTEROLOGY Vol. 100, No.5
SELECTED SUMMARIES
Therefore, the authors concluded that in utero transplacental exposure to HBeAg induced neonatal tolerance to both HBeAg and HBcAg. Comment. This study demonstrates that HBeAg-expressing mice, like other transgenic mice (Immunol Today 1989;10:53-57), are tolerant at the T -cell level to the protein product of their transgene. However, the unique structural interrelationship of HBeAg and HBcAg allowed the investigators in this study to make a number of interesting observations regarding HBcAg responses in such mice and in the nontransgenic offspring of HBeAg-expressing transgenic female mice. Thus, serum HBeAg contains 10 residues lacking in HBcAg and the C-terminal 34 residues of HBcAg are absent from HBeAg with the remainder of the primary amino acid sequence of the two polypeptides being identical (J Immunol 1983;130:29032912 and Proc Natl Acad Sci USA 1988;85:8405-8407). Whereas antibody responses to HBeAg and HBcAg are quite distinct, these antigens are highly cross-reactive in terms of T -cell responses. The authors have previously demonstrated that in the BI0.S mouse strain used in these studies, the dominant T-helper recognition site of both HBeAg and HBcAg is a common p120-131 peptide (J Immunol 1987;139:1223-1231). In the present studies in HBeAgexpressing BI0.S Tg-31e mice, the authors formally demonstrate the in vivo effects of such T-cell cross-reactivity. Thus, presumably because of both neonatal and lifelong exposure to endogenously synthesized HBeAg, such mice had defective T-proliferative responses not only to HBeAg but also to p120-131 and therefore to intact HBcAg. Moreover, while the relatively T-independent IgM response to HBcAg was preserved in such transgenic mice, major defects in T-helper cell-dependent IgG anti-HBcAg responses were apparent. Such results clearly relate to the fact that the immunodominant HBeAg and HBcAg T-cell epitopes recognized by this single inbred mouse strain are an identical peptide fragment common to both antigens. However, in previous studies by these authors (J Immunol 1987;139:1223-1231) in which multiple mouse strains have been surveyed, it has been shown that while the immunogenic peptide fragments capable of eliciting T-cell responses to HBeAg and HBcAg are different for mice with different MHC genes, such immunodominant epitopes are commonly observed to derive from regions of amino acid sequence shared between these two proteins. Thus, one can reasonably anticipate that even in outbred mice (or humans) similar T-cell cross-reactivity and hence cross-tolerance between HBeAg and HBcAg might be observed. Of perhaps even greater interest, the authors demonstrate that in utero exposure to HBeAg, presumably through the transplacental route, leads to a significant period of neonatal T-cell tolerance to HBeAg and HBcAg in non transgenic offspring of HBeAg-expressing transgenic female mice. Based on this finding, the authors propose that such a mechanism may explain the almost uniform development of a chronic carrier state following perinatal or postnatal HBV infection of human neonates born to HBeAg( +) mothers. For a variety of reasons this is an attractive hypothesis. The fact that human neonates readily respond to HBsAg vaccines seems to make this antigen a less likely tolerogen candidate. Some clinical data also exist to suggest that HBeAg crosses the human placenta (Hepatology 1986;6:369-373). Finally, unlike HBsAg and HBcAg, there appears to be no required role for HBeAg in HBV viral replication (J Virol 1987;61:3322-3325). Thus, the hypothesis as raised in this manuscript that HBeAg may represent a viral strategy to persist in the host after perinatal transmission is clearly a concept that is worthy of further consideration and study. D. L. THIELE, M.D.
LIVER DYSFUNCTION IN CYSTIC FIBROSIS-BENEFICIAL EFFECT OF BILE ACID TREATMENT Colombo C, Setchell KDR, Podda M, et al. (Departments of Pediatrics and Internal Medicine, University of Milan, Milan, Italy; the Institute of Analytical Chemistry, University of Messina, Messina, Italy; and the Clinical Mass Spectrometry Department, Children's Hospital Medical Center, Cincinnati, Ohio). Effects of ursodeoxycholic acid therapy for liver disease associated with cystic fibrosis. J Pediatr 1990;117:482-489 (September). This group from Milan, Italy, reports the results from a trial of bile acid therapy with ursodeoxycholic acid (UDCA) in patients with cystic fibrosis (CF). The patients were pretreated with supplemental taurine because of the taurine deficiency, secondary to malabsorption of taurine bile acid conjugates, known to be present in patients with CF. They then received UDCA therapy for 6 months in a dose of 10-15 mglkg. The authors followed standard liver "function" tests (LFTs) as well as serum albumin and prothrombin times. In addition, they investigated bile acid metabolism in these patients by characterizing the bile acids in both serum and bile. Serum bile acids were analyzed using competitive solid-phase enzyme immunoassays. Duodenal bile was sampled using the string test method and the proportions of total bile acids contributed by individual bile acids were defined using high-performance liquid chromatography (HPLC). Stool samples were assayed from 72-hour collections for both fat and bile acids. The patient population under study consisted of nine patients, all children, with liver disease as indicated by abnormal LFTs for 2 years. All patients had firm hepatomegaly. The biliary tree was patent in all as evidenced by normal hepatobiliary scintigraphy. In most patients, the most striking abnormality was in -y-glutamyl trans peptidase level, ranging from 70 to 484 (normal < 32). The patients were all relatively well, with none having a prolonged prothrombin time. Only one patient did not require pancreatic enzyme supplementation. One patient was known to have portal hypertension with esophageal varices. Liver biopsy was available in three and showed biliary cirrhosis in all three. All patients showed an improvement in LFTs during the 6 months of treatment. As anticipated, the biliary bile acids became enriched in UDCA with a decrease in the relative proportions of cholic and chenodeoxycholic acids. The serum level of UDCA rose, with no change in the serum levels of cholic and chenodeoxycholic acids. The authors point out that the relative enrichment of the bile acid pool in UDCA was less than that reported in adults with cholestatic disease treated with ursodeoxycholic acid. Similarly, the improvement in LFTs was less than that reported in cholestatic adults treated for other conditions. No change was observed in the stool fat excretion or, on average, in growth. The authors suggest that a larger dose of UDCA might be more beneficial and they conclude that the favorable results
SELECTED SUMMARIES
and> 65-year age groups. The largest number of patients (31%) were seen by general and family practitioners, followed in declining order by internists (20%) and pediatricians (15%). Only 4% of all these patients were seen by gastroenterologists. The impact of constipation on health care costs and morbidity are considerable. Fortunately, constipation results in fewer hospitalizations and deaths than other digestive disorders-25.5 million hospital days per year for peptic ulcers and gallbladder disease combined compared with 13.6 million days per year for constipation. However, in the United States alone, cathartics are prescribed for more than three million people yearly, with an estimated cost in the millions of dollars, and more than 200 million dollars are spent annually for over-the-counter laxatives UClin Gastroenterol1989; 11:525-536). The complications of constipation are particularly a problem for the elderly and bedridden population. Potential morbid problems include fecal impaction, fecal incontinence, stercoral ulcerations, megacolon with volvulus, ischemia or perforation, rectal prolapse, hemorrhoids, and rarely cardiac and cerebrovascular dysfunction (N Engl JMed 1989;321:658-661). In most older patients, constipation can be effectively treated by increasing dietary fiber and fluids or by adding a bulk-forming agent such as psyllium. However, an important subgroup requires additional laxative therapy which must be safe and suitable for long-term use. More and more the most commonly prescribed drugs for these patients are the osmotic agents particularly the nonabsorbable sugars such as lactulose and sorbitol. Those sugars have an osmotic laxative effect probably related to their conversion to organic acids by colonic bacteria and the resulting reduction in stool pH (Gut 1972;13:859-866). Their only side effects are bloating, flatulence, and diarrhea. Lactulose has been shown in several studies to be safe and effective for treating constipation in the elderly (Gut 1968:84-86; JAm Geriatr Soc 1978;26:236-239; J Clin Gastroenterol 1981;3(Suppl 1):23-28). This study is the first report showing the effectiveness and safety of sorbitol in a similar group of constipated patients. Unfortunately, the practical value oflactulose for treating constipation in the elderly has been limited by its high cost. The cost to the pharmacist for a 960-mL bottle of lactulose ranges from $30 to $50. In contrast, a similar quantity of sorbitol costs approximately $5. Sorbitol is approved by the Food and Drug Administration as a safe and effective laxative. However, it is usually considered only for specialized uses such as counteracting the constipating effects of potassium-binding resins rather than for general use in constipation. Why should this be? The authors of this article correctly suggest that the major determinants for prescribing practices are commercial marketing and the scientific literature (Am J Med 1982;73:4-8). Therefore, inexpensive generic drugs such as sorbitol would not be expected to appear in the advertising pages of various medical journals. Furthermore, the shortage of noncommercial funding for clinical research reduces the likelihood of generic agents appearing in the scientific literature (Lancet 1987;1:257258). If effective "Brand X" drugs are not to be overlooked, health care organizations and private foundations concerned with the rising cost of medical care and medications may need to become involved in supporting research into promising generic drugs. The authors of this article are to be thanked for reawakening our awareness to an inexpensive alternative to lactulose in the treatment of constipation in the elderly. Sorbitol may also be effective therapy for women, younger patients, or those who are bedridden but these studies need to be performed. Nevertheless, the use of sorbitol should save money for elderly patients and their provider organizations. Because the use of lactulose has been restricted as a cost control measure by many health care providers, sorbitol may also improve patient access to optimal therapy.
J. E. RICHTER, M.D.
1475
Editor's Note. Although this study was randomized and double blind, it was not placebo controlled. Moreover, baseline stool frequency (prior to laxatives) was not measured. Therefore, the efficacy of sorbitol and lactulose as a laxative in these ambulatory, elderly men cannot be determined from this study.
HEPATITIS e ANTIGEN AND NEONATAL IMMUNOLOGIC TOLERANCE TO HEPATITIS B Milich DR, Jones JE, Hughes JL, et a1. (Departments of Molecular Biology, Immunology, and Molecular and Experimental Medicine, Scripps Clinic and Research Foundation, La Jolla, California). Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero? Proc Natl Acad Sci USA 1990;87:6599-6603. To examine the tolerogenic potential of hepatitis B virus (HBV) antigens, the authors produced hepatitis B e antigen (HBeAg)-expressing transgenic mice and used a neonatal tolerance system to investigate the tolerogenic characteristics of the hepatitis B core antigen (HBcAg) and the HBeAg. The authors produced an HBeAg-expressing transgenic mouse lineage designated B10.S Tg-31e that contained a HBV DNA construct (subtype ayw, coordinates 1804-2804) with the complete pre core plus core open-reading frame linked to the mouse metallothionein I promoter. HBeAg was present at a level of 8-10 nglmL in the serum of these mice, but the sera were unreactive in an HBcAg-specific enzymelinked immunosorbent assy (ELISA). T cells from these B10.S Tg-31 transgenic mice were unresponsive to HBeAg, HBcAg, and the immunodominant peptide p120-131 common to both antigens. Furthermore, these transgenic mice did not produce anti-HBe spontaneously. When immunized with a particulate form of HBeAg expressing both HBe and HBc antigenicity, the B10.S Tg-31e mice produced a primary and secondary antibody response to HBcAg but only a very weak secondary antibody response to HBeAg. In vitro studies demonstrated that these transgenic mice produced a normal immunoglobulin M (lgM) response to HBcAg but a greatly reduced IgG response to this antigen, suggesting a defect in T -helper function for anti-HBc B-cell responses. A single neonatal injection with HBcAg was found to induce transient T-cell tolerance to HBcAg and the common HBc/HBe peptide p120-131. Additional studies were performed to assess the T-cell responder status of nontransgenic offspring of B10.S Tg-31e transgenic mice. Pregnant HBeAg-expressing transgenic mice were administered zinc in a dose that increased serum HBeAg levels to approximately 72 ngimL. T-cell proliferative responses of transgenic and non transgenic offspring were assessed at 6 weeks of age and compared with those of 6-week-old control mice. As expected, the transgenic offspring demonstrated significant tolerance to HBeAg and peptide fragments of HBeAg. Whereas nontransgenic littermates were not completely tolerant to these antigens, T-cell proliferative responses of these mice to HBeAg and the p120-131 peptide fragment common to HBeAg and HBcAg were intermediate to those seen in control and transgenic mice of the same age.
1476
GASTROENTEROLOGY Vol. 100, No.5
SELECTED SUMMARIES
Therefore, the authors concluded that in utero transplacental exposure to HBeAg induced neonatal tolerance to both HBeAg and HBcAg. Comment. This study demonstrates that HBeAg-expressing mice, like other transgenic mice (Immunol Today 1989;10:53-57), are tolerant at the T -cell level to the protein product of their transgene. However, the unique structural interrelationship of HBeAg and HBcAg allowed the investigators in this study to make a number of interesting observations regarding HBcAg responses in such mice and in the nontransgenic offspring of HBeAg-expressing transgenic female mice. Thus, serum HBeAg contains 10 residues lacking in HBcAg and the C-terminal 34 residues of HBcAg are absent from HBeAg with the remainder of the primary amino acid sequence of the two polypeptides being identical (J Immunol 1983;130:29032912 and Proc Natl Acad Sci USA 1988;85:8405-8407). Whereas antibody responses to HBeAg and HBcAg are quite distinct, these antigens are highly cross-reactive in terms of T -cell responses. The authors have previously demonstrated that in the BI0.S mouse strain used in these studies, the dominant T-helper recognition site of both HBeAg and HBcAg is a common p120-131 peptide (J Immunol 1987;139:1223-1231). In the present studies in HBeAgexpressing BI0.S Tg-31e mice, the authors formally demonstrate the in vivo effects of such T-cell cross-reactivity. Thus, presumably because of both neonatal and lifelong exposure to endogenously synthesized HBeAg, such mice had defective T-proliferative responses not only to HBeAg but also to p120-131 and therefore to intact HBcAg. Moreover, while the relatively T-independent IgM response to HBcAg was preserved in such transgenic mice, major defects in T-helper cell-dependent IgG anti-HBcAg responses were apparent. Such results clearly relate to the fact that the immunodominant HBeAg and HBcAg T-cell epitopes recognized by this single inbred mouse strain are an identical peptide fragment common to both antigens. However, in previous studies by these authors (J Immunol 1987;139:1223-1231) in which multiple mouse strains have been surveyed, it has been shown that while the immunogenic peptide fragments capable of eliciting T-cell responses to HBeAg and HBcAg are different for mice with different MHC genes, such immunodominant epitopes are commonly observed to derive from regions of amino acid sequence shared between these two proteins. Thus, one can reasonably anticipate that even in outbred mice (or humans) similar T-cell cross-reactivity and hence cross-tolerance between HBeAg and HBcAg might be observed. Of perhaps even greater interest, the authors demonstrate that in utero exposure to HBeAg, presumably through the transplacental route, leads to a significant period of neonatal T-cell tolerance to HBeAg and HBcAg in non transgenic offspring of HBeAg-expressing transgenic female mice. Based on this finding, the authors propose that such a mechanism may explain the almost uniform development of a chronic carrier state following perinatal or postnatal HBV infection of human neonates born to HBeAg( +) mothers. For a variety of reasons this is an attractive hypothesis. The fact that human neonates readily respond to HBsAg vaccines seems to make this antigen a less likely tolerogen candidate. Some clinical data also exist to suggest that HBeAg crosses the human placenta (Hepatology 1986;6:369-373). Finally, unlike HBsAg and HBcAg, there appears to be no required role for HBeAg in HBV viral replication (J Virol 1987;61:3322-3325). Thus, the hypothesis as raised in this manuscript that HBeAg may represent a viral strategy to persist in the host after perinatal transmission is clearly a concept that is worthy of further consideration and study. D. L. THIELE, M.D.
LIVER DYSFUNCTION IN CYSTIC FIBROSIS-BENEFICIAL EFFECT OF BILE ACID TREATMENT Colombo C, Setchell KDR, Podda M, et al. (Departments of Pediatrics and Internal Medicine, University of Milan, Milan, Italy; the Institute of Analytical Chemistry, University of Messina, Messina, Italy; and the Clinical Mass Spectrometry Department, Children's Hospital Medical Center, Cincinnati, Ohio). Effects of ursodeoxycholic acid therapy for liver disease associated with cystic fibrosis. J Pediatr 1990;117:482-489 (September). This group from Milan, Italy, reports the results from a trial of bile acid therapy with ursodeoxycholic acid (UDCA) in patients with cystic fibrosis (CF). The patients were pretreated with supplemental taurine because of the taurine deficiency, secondary to malabsorption of taurine bile acid conjugates, known to be present in patients with CF. They then received UDCA therapy for 6 months in a dose of 10-15 mglkg. The authors followed standard liver "function" tests (LFTs) as well as serum albumin and prothrombin times. In addition, they investigated bile acid metabolism in these patients by characterizing the bile acids in both serum and bile. Serum bile acids were analyzed using competitive solid-phase enzyme immunoassays. Duodenal bile was sampled using the string test method and the proportions of total bile acids contributed by individual bile acids were defined using high-performance liquid chromatography (HPLC). Stool samples were assayed from 72-hour collections for both fat and bile acids. The patient population under study consisted of nine patients, all children, with liver disease as indicated by abnormal LFTs for 2 years. All patients had firm hepatomegaly. The biliary tree was patent in all as evidenced by normal hepatobiliary scintigraphy. In most patients, the most striking abnormality was in -y-glutamyl trans peptidase level, ranging from 70 to 484 (normal < 32). The patients were all relatively well, with none having a prolonged prothrombin time. Only one patient did not require pancreatic enzyme supplementation. One patient was known to have portal hypertension with esophageal varices. Liver biopsy was available in three and showed biliary cirrhosis in all three. All patients showed an improvement in LFTs during the 6 months of treatment. As anticipated, the biliary bile acids became enriched in UDCA with a decrease in the relative proportions of cholic and chenodeoxycholic acids. The serum level of UDCA rose, with no change in the serum levels of cholic and chenodeoxycholic acids. The authors point out that the relative enrichment of the bile acid pool in UDCA was less than that reported in adults with cholestatic disease treated with ursodeoxycholic acid. Similarly, the improvement in LFTs was less than that reported in cholestatic adults treated for other conditions. No change was observed in the stool fat excretion or, on average, in growth. The authors suggest that a larger dose of UDCA might be more beneficial and they conclude that the favorable results