The dorzolamide/timolol combination versus timolol plus pilocarpine: patient preference and impact on daily life. United States Patient Preference Study Group. International Patient Preference Study Group.

PURPOSE: To compare the 2.0% dorzolamide/0.5% timolol fixed combination (COSOPT; Merck & Co., Whitehouse Station, NJ) to 0.5% timolol plus 2.0% pilocarpine given concomitantly, and to determine patient preference, tolerability, and impact on daily life in patients with elevated intraocular pressure (IOP). METHODS: Two multi-center, randomized, cross-over, observer masked studies were conducted, one in the United States (97 patients) and one in Europe (93 patients). The Comparison of Ophthalmic Medications for Tolerability questionnaire was used to assess patient preference and perception of side effects and activity limitations resulting from study medications. Intraocular pressure was measured before and 2 hours after the morning dose of study medication (hour 0 and hour 2). RESULTS: In both studies, among patients with a preference, the combination was preterred to timolol plus pilocarpine by a ratio of 4 to 1. The most commonly cited reason for this preference was side effects. Patients in both studies also reported that the combination interfered significantly less with daily life in terms of side effects and activity limitations. They also reported missing significantly fewer doses of study medication while taking the combination and being significantly more satisfied with it. The efficacy of these two treatments was not significantly different, based on IOP measurements at hour 0 and 2 hours after administration. Patients reported significantly more adverse events while receiving timolol plus pilocarpine in both studies, and in the U.S. study, significantly more patients discontinued therapy while receiving timolol plus pilocarpine than while receiving the combination. CONCLUSION: Compared with timolol plus pilocarpine, patients preferred the combination of 2% dorzolamide/0.5% timolol, and reported less interference in daily activities, better tolerability, and better compliance with therapy.

Comparison of the efficacy and safety of 2% dorzolamide and 0.5% betaxolol in the treatment of elevated intraocular pressure. Dorzolamide Comparison Study Group.

A multicenter, parallel-design, randomized, double-masked study was conducted to compare the efficacy and safety of 2% dorzolamide with those of 0.5% betaxolol in the treatment of elevated intraocular pressure (i.o.p). A total of 311 adults with ocular hypertension or open-angle glaucoma were randomly allocated to receive either 2% dorzolamide administered topically TID or 0.5% betaxolol administered topically BID plus placebo administered topically QD for 12 weeks. After the washout of previous ocular hypotensive drugs, patients with IOP > or = 23 mm Hg in at least one eye at 10 AM or 4 PM on study day 1 were randomly allocated to receive one of the study treatments. Throughout the study, IOP was measured 2 and 8 hours after instillation of study medication for the morning peak effect (hour 2) and afternoon trough effect (hour 8). After 12 weeks of therapy, the mean change in IOP was not significantly different between the dorzolamide and betaxolol treatment groups at hour 8 (-3.6 mm Hg in both groups) or hour 2 (-5.4 vs -5.3 mm Hg, respectively). The differences between treatments (and 95% CIs associated with these differences) in mean IOP changes from baseline were 0.02 mm Hg (-0.870 to 0.901) for hour 8 and -0.14 mm Hg (-0.959 to 0.685) for hour 2. The ocular adverse experience (AE) most frequently reported by patients was ocular burning and/or stinging, and the most frequently reported nonocular AEs were taste perversion, upper respiratory infection, and headache. Only the incidence of taste perversion was significantly different between treatment groups (14.6% for the dorzolamide group and 0.0% for the betaxolol group). Two percent of patients in each treatment group discontinued the study due to AEs. This study confirmed the similar IOP-lowering effect of 2% dorzolamide and 0.5% betaxolol. Both treatments were generally well tolerated, and their safety profiles were similar.

Antibody responses to capsular polysaccharide backbone and O-acetate side groups of Streptococcus pneumoniae type 9V in humans and rhesus macaques.

Streptococcus pneumoniae is responsible for high rates of pneumococcal bacteremia, meningitis, pneumonia, and acute otitis media worldwide. Protection from disease is conferred by antibodies specific for the polysaccharide (Ps) capsule of the bacteria. Of the four types of group 9 pneumococci, types 9N and 9V cause the most disease, and both types are included in the polyvalent pneumococcal vaccine. The type 9V capsule consists of repeating pentasaccharide units linearly arranged, with an average of 1 to 2 mol of O-acetate side chains per mol of repeat units, added in a complex pattern in which not all repeat units are alike. alpha-GlcA residues may be O-acetylated in the 2 (17%) or 3 (25%) position and beta-ManNAc residues may be O-acetylated in the 4 (6%) or 6 (55%) position. Under certain conditions, the O-acetate side chains are subject to oxidation, which results in subsequent de-O-acetylation of a significant number of the repeat units. This de-O-acetylation could adversely affect the efficacy of a vaccine containing the 9V Ps. A study was undertaken to compare the relative contributions of O-acetate and Ps backbone epitopes in the immune response to S. pneumoniae 9V type-specific Ps. In both an infant rhesus monkey model and humans, antibodies against the non-O-acetylated 9V backbone as well as against O-acetylated 9V Ps were detected. Functional (opsonophagocytic) activity was observed in antisera in which the predominant species of antibody recognized de-O-acetylated 9V Ps. We concluded that the O-acetate side groups, while recognized, are not essential to the ability of the 9V Ps to induce functional antibody responses.

Rickets screening in the preterm infant.

Although rickets in the preterm infant has decreased with improvements in care and nutrition, there continue to be infants at high risk for this disease. Early diagnosis and treatment can prevent fractures and other complications, such as decreased linear growth. Nurses must know which infants are at risk, must be aware of the appropriate screening tests, and must know how to customize care for at-risk neonates.

Immunogenicity of conjugate vaccines consisting of pneumococcal capsular polysaccharide types 6B, 14, 19F, and 23F and a meningococcal outer membrane protein complex.

In an effort to prepare pneumococcal (Pn) capsular polysaccharide (Ps) vaccines that would be immunogenic in infants, covalent conjugates were prepared for Pn types 6B, 14, 19F, and 23F. Each Ps type was covalently bound to an outer membrane protein complex from Neisseria meningitidis serogroup B and evaluated for immunogenicity in mice and infant monkeys. The conjugates induced specific anti-Ps antibody responses in mice and in infant rhesus and African green monkeys; a conjugate of 6B and outer membrane protein complex was immunogenic at Ps doses as low as 20 ng. Although low levels of the Pn group-common cell wall polysaccharide were present in all type-specific Ps preparations, anti-cell wall polysaccharide responses induced by covalent conjugates were < 1% of the total anti-Ps response after two doses of vaccine. In contrast, the anti-cell wall polysaccharide response of a noncovalent conjugate represented 41% of the anti-Ps response after two doses. Relative T-cell dependence, a requirement for the human target population of infants less than 18 months old, was demonstrated for all four Pn Ps conjugates in an athymic mouse model. Therefore, these Pn Ps-outer membrane protein complex conjugate vaccines are excellent candidates for evaluation in human infants.

Immunogenicity of a new Haemophilus influenzae type b conjugate vaccine (meningococcal protein conjugate) (PedvaxHIB).

Haemophilus influenzae type b is responsible for an estimated 15,000 to 20,000 cases of meningitis per year in the United States, mainly in children 2 months to 5 years old. The mortality rate from meningitis due to H influenzae type b infections ranges from 5% to 10%. Despite antibiotic treatment, up to 35% of survivors have permanent neurologic sequelae. In addition to meningitis, H. influenzae type b is responsible for other invasive infections, including epiglottitis, septicemia, cellulitis, septic arthritis, osteomyelitis, pneumonia, pericarditis, and otitis media; approximately 30,000 cases H influenzae diseases occur annually in the United States. The diseases peak in incidence between 6 and 12 months of age, with almost one half of the cases occurring before 1 year of age. About 75% of disease caused by H influenzae type b occurs in children younger than 24 months old. The incidence of disease is higher in children of certain groups, including blacks, Hispanics, Eskimos and Native Americans, young children attending day-care facilities, patients with asplenia or antibody-deficiency syndromes, and children of lower socioeconomic status. There is considerable evidence that antibody to the capsular polysaccharide (polyribosylribitol-phosphate [PRP] of H influenzae type b is protective.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemophilus influenzae type b conjugate vaccine (meningococcal protein conjugate) (PedvaxHIB): clinical evaluation.

Although systemic infections caused by Haemophilus influenzae type b occur worldwide, detailed epidemiologic data are available in but a few countries. The public health impact of morbidity, mortality, and serious sequelae from disease caused by H influenzae type b has stimulated the search for control strategies. In the United States now, active immunoprophylaxis is largely favored over treatment of prophylaxis with antibiotics. This preference stems from three major observations: that high mortality and morbidity persist despite the availability of potent antimicrobial agents, that antibiotic-resistant strains of H influenzae type b have emerged, and that implementation of antimicrobial prophylaxis on a large scale has been unsatisfactory. Moreover, universal vaccination has been projected as offering a higher economic benefit than other control strategies. A matter of more proximate importance, however, is the search for H influenzae type b vaccines that will confer protection to all age groups, including infants younger than 18 months of age and subpopulations specifically at risk for invasive disease caused by H influenzae type b. Haemophilus b conjugate vaccine (meningococcal protein conjugate), PedvaxHIB (PRP-OMPC), is a conjugate H influenzae type b vaccine developed at Merck Sharp & Dohme Research Laboratories that now is undergoing extensive clinical evaluation to assess its prospects for disease control when first administered in early infancy. This is an interim report of results obtained in studies conducted in diverse locations throughout the United States.

Septic plasma suppresses superoxide anion synthesis by normal homologous polymorphonuclear leukocytes.

In vitro exposure of normal O,Rh- PMN to plasma obtained from patients with septic shock results in inhibition of formyl-methionyl-leucyl-phenylalanine stimulated superoxide anion (O2.-) production by 40%. Although all reaction velocities and extent of reaction at 5 min were suppressed, neither lag time preceding O2.- production nor duration of initial velocity linearity was affected. No such inhibition was noted when plasma from healthy controls or nonseptic critically ill patients was utilized in the reaction. These results demonstrate that neutrophils are not only a cause, but also a target of the septic shock host inflammatory response.

Effects of arginine-free diet on urea cycle enzymes in young and adult ferrets.

Young ferrets develop hyperammonemia soon after eating an arginine-free diet, whereas adult ferrets do not develop hyperammonemia after an identical treatment. Earlier reports indicate that young or adult rats do not develop hyperammonemia and encephalopathy after a single meal of an arginine-free diet. The effects of a single feeding of an arginine-free diet on the urea cycle enzyme activities in the liver of young and adult ferrets is reported. Ornithine carbamyl transferase, carbamyl phosphate synthetase and ornithine aminotransferase activities in the livers of adult ferrets were significantly higher than those in the livers of young ferrets. A single meal of an arginine-free diet did not alter the urea cycle enzyme activities in the liver of young or adult ferrets. The levels of urea cycle enzymes in the liver and kidney of young ferrets were comparable to those in rat liver and kidney. The results suggest that the hyperammonemia observed in young ferrets following a single meal of an arginine-free diet may not be due to the deficiency of enzyme activities.

Structure-function relationships for the interleukin 2 receptor: location of ligand and antibody binding sites on the Tac receptor chain by mutational analysis.

The Tac protein plays a role in high- and low-affinity interleukin 2 (IL-2) receptors. A mutational survey of this molecule identified several small segments in which the binding of IL-2 was particularly sensitive to amino acid substitutions. Two of the segments (residues 1-6 and 35-43) located in the exon 2-encoded region of the molecule overlapped the apparent binding sites of three monoclonal antibodies (anti-Tac, GL439, and H31) that block high- and low-affinity Tac-IL-2 interactions, thus supporting the hypothesis that these segments of the protein are at or near sites of receptor-ligand contact. In contrast, the apparent binding sites of antibodies (Hiei and H47) that selectively inhibit high-affinity IL-2 binding were mapped to a distinct location (residues 158-160) within the region encoded by exon 4 of the Tac gene. Since high-affinity receptors consist of a heterodimer of Tac and a second ligand-binding protein (p70), this portion of the Tac molecule may be involved in the interaction between the two receptor subunits. As expected, the binding sites of noninhibitory antibodies (7G7/B6, residues 140-144; H48, residues 170-211) did not overlap those segments in which IL-2-binding mutants were observed. These results provide a preliminary correlation of structure and function for the Tac protein that should prove useful in evaluating detailed models of the IL-2-receptor complex.

Structure-function relationships for the IL-2 receptor system. V. Structure-activity analysis of modified and truncated forms of the Tac receptor protein: site-specific mutagenesis of cysteine residues.

IL-2R on activated lymphocytes contain the Tac protein. As part of an effort to characterize this molecule, we examined the structure-activity relationship for each of its 12 Cys residues. A preliminary map of intramolecular disulfide bonding was derived by analysis of cystine-linked enzymatic fragments of the Tac protein. The results indicated that disulfide bonds linked Cys-3 with Cys-147, Cys-131 with Cys-163, and Cys-28,30 with Cys-59,61. The contribution of the Cys residues to an active protein conformation was tested by site-specific mutagenesis, followed by expression of the modified molecules in murine L cells. The results indicated that Cys-192 and -225 could be replaced without affecting ligand binding. In contrast, modification of any of the other 10 Cys residues, either singly or in combinations corresponding to the predicted disulfide bonds, greatly reduced the ability of the corresponding protein to bind IL-2 or either of two mAb (anti-Tac and 7G7/B6) which recognize the Tac protein. Each of the latter mutations also interfered with the molecule's post-translational modification and cell-surface expression. Consistent with these findings, transfection of the L cells with vectors containing truncated Tac cDNA inserts resulted in secretion of Tac fragments capable of ligand binding when the polypeptide chains terminated after Cys-163 (the 10th Cys residue in the full length molecule), but resulted in inactive fragments of Tac which were poorly secreted when they terminated before Cys-163. These findings emphasize the remarkable sensitivity of the active conformation of the Tac molecule to each of the postulated intramolecular disulfide bonds.

Failure of L-carnitine to protect mice against ammonia toxicity.

Recent reports indicate that intraperitoneal administration of L-carnitine protects mice from ammonia toxicity. We found that mice injected with L-carnitine and subsequently challenged with ammonium acetate succumb as readily as mice injected with saline and the ammonium acetate. Mice pretreated with L-carnitine exhibited higher levels of liver ammonia than the saline-pretreated control mice. The ammonia and urea levels in serum and brains were similar in two groups. Our findings are in contrast to those reported previously and therefore warrants further investigation before L-carnitine can be considered as a drug to alleviate hyperammonemia in humans.

Interleukin 2 binding molecule distinct from the Tac protein: analysis of its role in formation of high-affinity receptors.

Interleukin 2 (IL-2) receptors on activated T cells exist in high- and low-affinity configurations, both of which share a ligand-binding component known as the Tac protein. Although almost all binding of IL-2 to such cells was inhibited by an antibody to Tac, the predominant component of binding on the natural killer (NK)-like cell line YT was resistant to this reagent. The ligand-binding component on YT cells also differed from Tac in its affinity constant (Kd approximately 8.2 X 10(-10) M vs. Kd approximately equal to 1.1 X 10(-8) M for low-affinity Tac sites) and in its susceptibility to inhibition by certain antibodies to IL-2. When the YT cells were stimulated in a manner that induced expression of the Tac protein, the IL-2 binding sites were converted to a high-affinity configuration (Kd approximately 1.8 X 10(-11) M). Thus, the original binding component on unstimulated YT cells appeared to combine with Tac and IL-2 to produce a high-affinity receptor complex. Use of bifunctional crosslinking agents following ligand binding to unstimulated YT cells yielded covalent IL-2-receptor complexes of 83 and 90 kDa. These complexes were similar in size to those derived from high-affinity receptors on activated T cells and shared a similar fragmentation pattern upon proteolysis. These results demonstrate the existence of a second IL-2 binding component in addition to the Tac protein and suggest that this component combines with Tac and IL-2 to form high-affinity receptor sites.

Structure-function relationships for the IL 2-receptor system. II. Localization of an IL 2 binding site on high and low affinity receptors.

The ligand-binding component of high and low affinity IL 2 receptors is a 55,000 m.w. glycoprotein termed Tac. Correlating the structure and function of this molecule should provide insight into the mechanism of IL 2-initiated signal transduction and the structural basis for high and low affinity receptor forms. As a first step in this process, various approaches were used to localize the IL 2 binding region of the Tac molecule. Antibodies prepared to synthetic fragments of Tac were tested for their ability to interfere with IL 2 binding and bioactivity. The results delineated segments in the C-terminal portion of the molecule which appeared to be distal to the ligand binding site. In a more direct approach, radioiodinated IL 2 was cross-linked to high and low affinity receptors, and the resulting complexes were subjected to mild tryptic digestion. Consistent with the antibody data, the IL 2 remained covalently associated with an N-terminal tryptic fragment which apparently consisted of residues 1-83 of the Tac protein. These results suggest that the N-terminal region of the Tac molecule contains important contact sites for ligand-receptor interaction.

High and low affinity receptors for interleukin 2: implications of pronase, phorbol ester, and cell membrane studies upon the basis for differential ligand affinities.

Cellular binding sites for IL 2 exist in two forms which differ with respect to their apparent affinity for the factor. The present studies were designed to evaluate various models for the difference. Receptor-mediated internalization and covalent receptor-ligand coupling were discounted as explanations on the basis of ligand binding and elution studies on permeabilized cells and cell membranes. Phosphorylation of the receptor during activation of protein kinase C failed to modulate the ratio of high and low affinity sites, demonstrating that it also did not provide a potential mechanism. Selective destruction of low affinity receptors with pronase, on the other hand, indicated that the two forms of binding sites differed significantly in their cell surface structure. Either the two types of receptor consist of distinct molecules or the conformation of the high affinity binding sites renders them more resistant to proteolysis. Antibody inhibition studies revealed that the high affinity receptors remaining after protease treatment and their low affinity counterparts both utilized the same ligand-binding component. Thus, this result ruled out the possibility of two totally distinct receptor structures. Together, the findings support the hypothesis that other membrane components modify the conformation of the ligand-binding polypeptide to confer a high affinity protease-resistant configuration.

Human argininosuccinate synthetase minigenes are subject to arginine-mediated repression but not to trans induction.

The human argininosuccinate synthetase locus is subject to metabolite-mediated repression by arginine in some cultured cell lines. To gain insight into the mechanism underlying this regulation, chloramphenicol acetyltransferase (CAT) minigenes under the transcriptional control of the human argininosuccinate synthetase promoter were constructed and tested for regulation. When the minigenes were introduced into RPMI 2650 cells, a human cell line that shows sixfold regulation of the argininosuccinate synthetase gene, CAT expression was repressed three- to fivefold when arginine was present in the culture medium. A minigene containing only 149 base pairs of 5'-flanking sequence was expressed at similar levels and regulated to the same degree as one having approximately 3 kilobases of 5'-flanking sequence. Therefore, the cis-acting sequences required for the arginine-mediated repression are likely to be located within the region of the transcription initiation site. The arginine-mediated repression of the CAT minigenes was not observed in canavanine-resistant variants of RPMI 2650 cells, and therefore they showed the appropriate cell-type specificity. Cultured cells having 200-fold-increased levels of argininosuccinate synthetase can be selected by growth in medium containing the arginine analog canavanine. It was previously demonstrated that the increased expression of argininosuccinate synthetase in canavanine-resistant human lymphoblasts was due to a trans-acting mechanism. To gain further support for a trans-acting mechanism, we tested our CAT minigenes for the trans induction in canavanine-resistant variants of RPMI 2650 cells. Transfection of the CAT minigenes into RPMI 2650 cells and canavanine-resistant variants of this cell line yielded no difference in transient CAT expression. Furthermore, cloned canavanine-resistant variant cells having integrated copies of the CAT minigenes expressed CAT at similar levels as compared to the parental cell lines. Since these cell lines do exhibit arginine-mediated repression of CAT but not trans induction, these data indicate that the argine-mediated repression is a regulatory event that occurs independently of the trans induction.

Stable expression of cDNA encoding the human interleukin 2 receptor in eukaryotic cells.

Human interleukin 2 (IL-2) receptor cDNA derived from HUT 102B2 cells was stably expressed in murine L cells. These L cell transfectants (a) displayed surface receptors of the aberrant size of the IL-2 receptors on HUT 102B2 cells, (b) did not respond to exogenous IL-2 with augmented proliferation, and (c) expressed low affinity but not high affinity receptors for IL-2.

Low and high affinity cellular receptors for interleukin 2. Implications for the level of Tac antigen.

Interleukin 2 promotes proliferation of T cells by virtue of its interaction with a high-affinity cell surface receptor. This receptor is a 55,000 mol wt glycoprotein that is also recognized by the murine monoclonal antibody, anti-Tac. Quantitative binding studies with radiolabeled IL-2 and anti-Tac, however, initially indicated far more antibody binding sites per cell than IL-2 binding sites. Extension of the IL-2 binding analysis to concentrations several thousand-fold higher than that necessary for the T cell proliferative response demonstrated the existence of a class (or classes) of low-affinity IL-2 binding sites. Inclusion of the low-affinity IL-2 binding greatly reduced the quantitative discrepancy in the ligand binding assays. That the low-affinity binding, as well as the high-affinity interaction, was associated with the Tac molecule was indicated by the finding that the antibody could substantially or totally block the entire spectrum of IL-2 binding and by the finding that IL-2 could in turn block all radiolabeled anti-Tac binding. The low-affinity sites were found on activated T cells, several human and murine T cell lines and two examples of Tac-positive B cells. The physiological role of the low-affinity IL-2 binding sites and the molecular changes in the Tac protein that give rise to the affinity differences remain open to investigation.

A cell kinetic method for the mitotic selection of treated G2 cells.

G2 cells treated with 150 rad X-radiation were isolated from a monolayer culture of exponentially growing Chinese hamster ovary (CHO) cells by a combination of 125Iododeoxyuridine ([125I]UdR)-induced blockade of S-phase cell progression, treatment and mitotic selection (125I-TMS technique). Once the rate at which cells were selected from a small window in mitosis was established (Schneiderman et al., 1972), the cells were exposed to 10 microCi/ml, carrier-free [125I]UdR for 10 min immediately before treatment with 150 rads X-radiation. After X-irradiation the cells located later in the cell cycle than the X-ray-induced division delay transition point (TPx), at or just prior to prophase, progressed without delay and were selected during the next 50 min (Walters & Petersen, 1968; Schneiderman et al., 1972). The G2- and S-phase cells, located prior to the TPx, sustained a transitory delay and resumed progression into mitosis only after recovery from the radiation insult (Terasima & Tolmach, 1963). However, S-phase cells having incorporated [125I]UdR during the pulse label were prevented from entering mitosis (Schneiderman & Hofer, 1980) and only the X-ray-treated G2 cells resumed progression into mitosis and were selected.