Expression and purification of biologically active domain I of the human polymeric immunoglobulin receptor.

Previous studies using proteolytic fragments and synthetic peptides have indicated that domain I of human polymeric immunoglobulin receptor (PIgR) is necessary for ligand binding. The expression in E. coli, and subsequent IgM-affinity purification of domain I of human PIgR is described. The recombinant domain I protein (rDI) was similar in structure to native SC domain I in that it bound specifically to MAb 6G11, an antibody which recognizes a critical portion of the PIg binding site in domain I. The biological activity of rDI was indicated by high affinity binding to PIgA (Kd = 1.6 x 10(-7) M) and IgM (Kd = 5.1 x 10(-7) M). Domain I of human SC is therefore sufficient for binding to PIg.

Thyrotropin (TSH) interacts with multiple discrete regions of the TSH receptor: polyclonal rabbit antibodies to one or more of these regions can inhibit TSH binding and function.

As a means of identifying functional regions of the TSH receptor (TSHr), we immunized four rabbits with recombinant extracellular TSHr (ETSHr) protein and systematically evaluated their antibody response. The antibody response was characterized by testing serial serum samples for immunoglobulin G (IgG) against ETSHr protein and 26 synthetic peptides which span the entire ETSHr. Sera were also tested for their ability to block TSH binding to native TSHr. All four rabbits developed high serum IgG titers (>1:100,000) to ETSHr. None of the rabbits developed significant IgG titers against 11 of the peptides, but each showed persistent high titers against several of the others. After multiple inoculations of antigen, sera from 3 rabbits showed significant ability to block TSH binding. Based on the ability of peptides to reverse this blocking activity, we identified 3 regions of the TSHr (i.e. amino acids 292-311, 367-386, and 397-415) through which antibodies can block TSH binding. Moreover, antibodies purified on either peptide 292-311 or peptide 367-386 affinity columns could block both TSH binding and TSH-mediated activation of thyroid cells in culture. These studies show ETSHr protein is sufficient to induce production of functionally relevant antibodies. Furthermore, we have identified several sites on the TSHr through which antibodies can inhibit TSH binding, thus leading to identification of several potential TSH-binding regions.

Pharmacokinetics, cortisol release, and hemodynamics after intravenous and subcutaneous injection of human corticotropin-releasing factor in humans.

OBJECTIVE: Two clinical trials investigated the pharmacokinetics of human corticotropin-releasing factor (hCRF), resulting cortisol release, and associated hemodynamic changes. METHODS: In a 3 x 3 Latin square design, subjects were randomized to receive a single dose of 5 microg x kg(-1) hCRF as a 10-minute intravenous infusion, a 180-minute infusion, and a subcutaneous injection in separate study sessions 7 days apart. Twelve additional subjects obtained a subcutaneous dose of either 300, 600, or 1200 microg hCRF on 3 consecutive days. Noncompartmental and compartmental pharmacokinetic analysis was performed. Hemodynamic response was characterized with use of pharmacodynamic models. RESULTS: The volume of distribution at steady state was 9.81 +/- 3.0 and 15.61 +/- 2.9, and the clearance was 256 +/- 40 mL x min(-1) and 345 +/- 90 mL x min(-1) for the 10-minute and 180-minute intravenous infusion, respectively (P < .05). Corresponding elimination half-life was 45 +/- 7 minutes and 37 +/- 10 minutes. Two-compartment and 1-compartment models adequately described the 10-minute and 180-minute infusions, respectively. The bioavailability of hCRF after subcutaneous administration was 67% +/- 17%. Apparent clearance remained unchanged for different subcutaneous doses. Peak plasma cortisol concentrations were similar after subcutaneous and intravenous administration of hCRF. Repetitive administration of hCRF did not result in accumulation but produced a reduced plasma cortisol response. A sigmoidal model related plasma hCRF concentrations to increase in heart rate (maximum, 39 beats x min(-1)). The relationship between the modest decrease in diastolic blood pressure and plasma hCRF concentrations was linear. CONCLUSION: The pharmacokinetics of intravenously administered hCRF were nonlinear, but apparent clearance was constant for various subcutaneous doses. An excellent bioavailability and preserved bioactivity make the subcutaneous route an attractive choice. Repetitive administration of hCRF probably caused tolerance of the cortisol response.

Progressive dystonia masking ataxia in ataxia-telangiectasia.

A 10-year-old boy with ataxia-telangiectasia had severe progressive dystonic posturing that masked the ataxia until treatment relieved the dystonia. A younger sister had mor classical neurologic manifestations of the disease. However, both children had telangiectasia, immunologic abnormalities, and other features of ataxia-telangiectasia. The pathologic changes that have been found in the basal ganglia at autopsy and the occurrence of choreoathetosis, oculomotor disturbances, and now dystonia indicate that the function of the basal ganglia in patients with ataxia-telangiectasia is abnormal. Children who have basal ganglial abnormalities should be studied for ataxia-telangiectasia.

Disseminated cryptococcus in an infant with severe combined immunodeficiency.

Opportunistic infections, often fatal, are frequent concomitants of congenital and acquired immunodeficiencies. The authors report a case of fatal cryptococcosis in a 5-month-old male infant with severe combined immunodeficiency. The anatomic distribution of cryptococcal lesions suggests that the terminal small intestine, as well as the lower respiratory tract, may serve as a portal of entry for this organism.

Human herpesvirus 8 DNA sequences in blistering skin from patients with pemphigus.

BACKGROUND: Human herpesvirus 8 (HHV-8) has been detected in Kaposi sarcoma (KS) and other lesions in patients both seropositive and seronegative for the human immunodeficiency virus (HIV). Kaposi sarcoma has been reported to develop in a disproportionate number of patients with pemphigus. Since HHV-8 is so strongly associated with KS, we wondered whether HHV-8 is present in pemphigus lesions from patients without KS or HIV infection. Pemphigus lesions and skin from healthy individuals were coded in a blinded fashion. Tissue-extracted DNA was tested using polymerase chain reaction, Southern blot hybridization, and automated sequencing of the polymerase chain reaction products for the presence of HHV-8 DNA. Six patients had pemphigus foliaceus, 6 had pemphigus vulgaris, and 2 had KS; 10 healthy individuals were used as controls. All 24 patients were HIV seronegative. OBSERVATION: Lesional skin from 4 of the 6 patients with pemphigus vulgaris, all 6 of the patients with pemphigus foliaceus, and both positive controls (KS) tested positive for HHV-8 DNA. Furthermore, the HHV-8 DNA sequences for KS330(233) differed between all 6 DNA specimens from pemphigus foliaceus, while 3 of the 4 DNA specimens from pemphigus vulgaris were identical. However, HHV-8 DNA was absent in all normal human skin analyzed. CONCLUSIONS: This report expands the spectrum of lesions found to contain HHV-8 DNA sequences and suggests that HHV-8 might have trophism for pemphigus lesions.

The effect of silicone ocular surgical devices on serum IgG binding to silicones.

PURPOSE: To determine whether silicone materials used in retinal detachment repair and cataract surgery increase serum IgG binding to silicone and identify correlations with complications of ocular surgery. METHODS: Serum from 49 patients who had ocular surgery using silicone materials was examined. Patient groups included scleral buckling (n = 25), silicone oil tamponade (n = 3), scleral buckling and silicone oil tamponade (n = 9), and silicone lens implants after cataract extraction (n = 12). Convalescent samples for all patients and preoperative samples from 19 patients (18 scleral buckling and one silicone oil tamponade) were examined. Postoperative complications were monitored for up to 108 months (mean, 10.7 months; mode, 1.5 months; range, 1 to 108 months). Samples were evaluated for the extent of IgG binding to silicones using a micromodification of a previously described enzyme-linked immunosorbent assay method. RESULTS: In 19 patients, IgG binding levels in preoperative samples were 21 arbitrary units (AU) or less. Of the 25 buckling patients, one developed complications; however, in all patients the postoperative levels of IgG binding to silicone were low (2.2 to 20.0 AU). Although four silicone lens patients developed mild complications, none displayed postoperative IgG binding levels of greater than 20 AU. Three patients who underwent both scleral buckling and silicone oil tamponade developed complications; one of these patients, who was also noted to have systemic connective tissue disease, had a significant elevation in postoperative serum IgG binding to silicone. CONCLUSIONS: Statistically significant elevations of serum IgG binding to silicone were noted postoperatively in only one patient who had a systemic connective tissue disease. The complication rate and frequency of enhanced serum IgG binding to silicone was low, making correlations to surgical complications difficult. Examination of matched samples suggested that if ocular exposure to silicone implants enhances the level of serum IgG binding to silicones, it must be a rare event that should not alter the clinical use of these important devices.

Long-term safety of MorphiDex.

More than 2200 subjects were enrolled in the MorphiDex (MS:DM) development program, with a 1:1 (weight:weight) ratio of morphine sulfate (MS) to dextromethorphan hydrobromide (DM). Of the 1400 subjects exposed to MorphiDex, more than 350 subjects were treated for at least 6 months, and over 200 subjects were treated for a year or longer. The clinical population comprised an approximately equal number of men (46.2%) and women (53.8%), ranging in age from 16 to 96 years, and mostly Caucasian (91.8%). The most frequent (54.8%) daily dose of MorphiDex for subjects enrolled in the clinical program was 120 mg or less. Slow DM metabolizers took significantly lower daily doses of MorphiDex than rapid metabolizers without a significant difference in the incidence of adverse events. Plasma bromide concentrations were low and showed a wide margin of safety for both slow and rapid DM metabolizers. There were no clinically significant treatment-related changes in clinical laboratory tests, neurological examinations, or vital signs. The most common adverse events seen in the multiple dose controlled studies were nausea, dizziness, vomiting, somnolence, constipation, confusion, asthenia, headache, and pruritus. With long-term treatment, the prevalence of adverse events was greatest during the first month of MorphiDex exposure and then decreased over time. The incidence of constipation remained fairly constant over time.

Facilitating faculty development and research through critical review of grant proposals and articles.

BACKGROUND: In 1983 the Department of Pediatrics at the University of Texas Medical Branch at Galveston established a faculty development program to address faculty needs for continuing education and improved resources for research. At first a part-time coordinator was hired; then, in 1985, a full-time, faculty-level science communicator provided help with strategic planning of projects and intensive review of grant proposals and journal articles. Faculty participation in the program was voluntary. METHOD: Pre- and post-intervention data for 1983-1992 included numbers of faculty using the program, faculty evaluations of the program, grant dollars awarded, counts of grant submissions and awards, and numbers of published articles. RESULTS: The review services were used heavily for grant proposals (75% of the department's proposals), but were used lightly for research articles (18% of publications). Grant funding quadrupled from 1983 to 1988; although funding peaked in 1988, it thereafter remained at three to four times the 1983 level. In contrast, the mean number of publications per faculty per year dropped between 1983 and 1990. CONCLUSION: The program provided valuable assistance to the faculty in writing grant proposals, and it helped to generate critically needed resources. However, the program's failure to increase the publication productivity of the faculty suggests that despite financial pressures, similar programs should use their influence and resources to promote a balance between scholarly publication and grant acquisition.

Therapy of rheumatoid arthritis with mycophenolate mofetil.

Mycophenolate mofetil, a pro-drug for mycophenolic acid, is an investigational immunosuppressive compound that is being developed for the treatment of rheumatoid arthritis (RA). The drug and its primary metabolite, mycophenolic acid, inhibit the de novo pathway of purine biosynthesis and have greater anti-proliferative effects on lymphocytes than on other rapidly dividing cells. Significant clinical improvement has been seen in many mycophenolate mofetil-treated RA patients who have been refractory to treatment with a variety of disease-modifying anti-rheumatic drugs (DMARDs). Treatment with mycophenolate mofetil reduces rheumatoid factor titres, immunoglobulin (IgG, IgM, and IgA) levels, and the total number of T cells (CD2) in RA patient peripheral blood; in addition, lymphocyte mitogen responses are inhibited and delayed hypersensitivity skin test reactivity is decreased. A dose of 2 g daily is more effective than lower doses, including several pulsing regimens. The most frequent adverse events reported by patients on mycophenolate mofetil are gastrointestinal, mainly nausea, vomiting, abdominal pain, and diarrhea. RA studies have demonstrated no clinically significant nephrotoxicity, hepatotoxicity, or bone marrow toxicity attributable to mycophenolate mofetil.

Immunologic safety of ibuprofen in rheumatoid arthritis: preliminary evidence.

Some evidence indicates that ibuprofen and other prostaglandin synthetase inhibitors may have the potential for cellular immune enhancement in addition to their anti-inflammatory activity. If this is true, treatment of rheumatoid arthritis, a disorder of presumed autoimmune pathogenesis, would present a dilemma. These agents are widely used in rheumatoid arthritis for their anti-inflammatory effects. If they are found to enhance cellular immune function, however, the disease might be stimulated over the long term, rather than suppressed. Preliminary evidence from four patients with rheumatoid arthritis show that oral ibuprofen had no significant immunologic effect during sequential "on" and "off" cycles, as assessed by the following measures: delayed hypersensitivity skin testing; lymphocyte transformation to mitogen (phytohemagglutinin) or specific antigen (Candida albicans); T-cell subsets, as determined by monoclonal antibody techniques; or production of the lymphokine, human immune interferon, in response to phytohemagglutinin or to staphylococcal enterotoxin A. Early evidence, therefore, suggests that oral ibuprofen therapy may be 'immunologically safe' in patients with rheumatoid arthritis, but investigations of large series of patients also assessing local immune reaction in diseased joints may be necessary for confirmation.

Primary deficiencies in humoral immunity.

Patients with primary disorders of B lymphocytes and immunoglobulins usually display increased susceptibility to bacterial infections but atopic, autoimmune, and malignant disorders are also more common in these patients. The spectrum of these disorders ranges from a virtual absence of B cells and immunoglobulins to selective deficiencies of immunoglobulin subclasses. The diagnosis is dependent upon the demonstration of the immunologic deficits by specialized laboratory procedures which include the quantitation of immunoglobulins, the formation of antibodies in vivo and in vitro and the demonstration of B cells in the tissues or the peripheral blood. There are five major points in the management of these patients: (1) the delineation of the immunologic defects by laboratory testing, (2) the use of parenterally injected human immunoglobulins, (3) the rapid identification of infecting organisms and the prompt institution of appropriate antibiotic therapy, (4) screening the family for immunodeficiency in those cases which are of genetic origin, and (5) genetic counseling.

A variant of toxic shock syndrome. Clinical, microbiologic, and autopsy findings in a fatal case.

A 16-year-old boy had toxic shock syndrome (TSS); Staphylococcus aureus bacteremia developed 11 hours prior to his death, which was three days after onset of the illness. The isoelectric focusing pattern of the staphylococcal isolate differed from both non-TSS and classic TSS S aureus isolates. Anatomic findings suggest three pathogenetic mechanisms: (1) immune complex-associated pulmonary microangiitis and vasculitis in the skin and skeletal muscle; (2) parenchymal cell "microvesicular" fatty metamorphosis in the liver, myocardium, renal tubules, and pancreas, and (3) pancarditis.

Anti-inflammatory systems in human milk.

Human milk is characterized not only by a complex host defense system that prevents the colonization and proliferation of common microbial pathogens that may pervade the alimentary tract and respiratory tract of the infant but also by a paucity of inflammatory agents and an array of anti-phlogistic factors. Clinical observations support the notion that the protection provided by human milk involves not only antimicrobial factors, but also anti-inflammatory agents. The major anti-inflammatory agents include enzymes that degrade mediators of inflammation, anti-proteases, lysozyme, lactoferrin, secretory IgA and a number of antioxidants including cysteine, ascorbate, alpha-tocopherol, and beta-carotene. It is pertinent that most of these factors are either absent or poorly represented in cow's milk or other artificial feedings that substitute for breast feeding and that the attainment of adult serum levels of some of these antioxidants in early infancy is dependent upon breast feeding. It may be that the provision of these antioxidants may help to protect the recipient's developing immunologic system which is quite susceptible to oxidant damage. The absence of breast feeding will thus deprive the infant of valuable protection against common enteric-respiratory disorders and their inflammatory consequences. It should be pointed out that the protective systems in human milk including the anti-inflammatory components may not be completely delineated, and that little is known of the in vivo fate of the factors and precisely how they protect the recipient. Those questions should form the basis of important research in the next decades.

Host defenses: development and maternal contributions.

During the intrauterine period, the human immunologic system develops through a complex but orderly series of events. The functional capacity of the system remains incomplete, not only during prenatal life but also through much of infancy. Many of the factors not produced by the fetus or infant are provided by the mother. Systemic immunity is augmented by specific IgG antibodies from the placenta and mucosal immunity by a wide array of defense agents from human milk including sIgA antibodies, lactoferrin, lysozyme, other soluble factors with antimicrobial properties, and specifically adapted leukocytes. It appears that the defense of the infant and the maternal contribution to that defense are geared to protect principally by noninflammatory mechanisms. Although much has been discovered about the ontogeny of the human immunologic system and the maternal contributions to this immunity, much remains to be learned about the molecular controls of the system, the fate of the transported maternal factors, feedback mechanisms between the immunologic systems of the mother and infant and the precise effects of maternal factors upon the infant. Answers to these questions may lead to the development of immunizing agents which are better suited to the infant, mucosal immunogens fashioned to stimulate the production of protective SIgA antibodies in human milk, the provision of defense factors for serious infections in young infants, and ways to enhance the maturation of the immunologic system of the infant when that is desirable.