Identification of a Hevea brasiliensis latex manganese superoxide dismutase (Hev b 10) as a cross-reactive allergen.

BACKGROUND: Cross-reactive allergens play an increasingly important role in latex allergy in complicating both the diagnosis and time course of allergic symptoms. Manganese superoxide dismutase (MnSOD), a ubiquitous protein of prokaryotic and eukaryotic organisms, was described as a cross-reactive allergen in Aspergillus fumigatus. Little information is available on the importance of this pan-allergen in Hevea brasiliensis latex. The aim of this study was to clone and express MnSOD from H. brasiliensis latex, and to obtain the soluble and immunologically active recombinant allergen for diagnosis of latex allergy and to investigate possible cross-reactivities with the structurally related A. fumigatus and human MnSODs. METHODS: A complementary DNA coding for Hevea latex MnSOD was amplified by PCR. The recombinant protein was produced in Escherichia coli with an N-terminal hexahistidyl tag. Enzymatic activity of the recombinant protein was determined using an enzyme assay for SODs. IgE immunoblotting and IgE inhibition assays were performed to characterize the recombinant allergen and its cross-reactivity. RESULTS: A Hevea latex MnSOD consisting of 206 amino acid residues was cloned and expressed in E. coli. The allergen was designated Hev b 10. The recombinant protein was enzymatically active, indicating the correct folding of the protein. In immunoblots, latex- as well as A. fumigatus-allergic patients revealed IgE binding to recombinant (r)Hev b 10. Cross-reactivity to Asp f 6, the MnSOD from A. fumigatus, and human MnSOD was determined by inhibition of IgE binding to these MnSODs by rHev b 10. CONCLUSIONS: Hev b 10 is a new cross-reactive allergen of H. brasiliensis which belongs to the 'latex-mold' group of latex allergens. Furthermore, it is a candidate for primary sensitization in patients allergic to the pan-allergen MnSOD.     

Antibody response to low-molecular-weight antigens of Aspergillus fumigatus in allergic bronchopulmonary aspergillosis.

Sera from patients with allergic bronchopulmonary aspergillosis (ABPA) or aspergilloma and normal sera were analyzed for specific antibodies by Western (immuno-) blotting with Aspergillus fumigatus antigens transferred electrophoretically onto polyvinylidene difluoride membranes. Western blot analysis demonstrated consistent reactivity of low-molecular-weight A. fumigatus antigens against ABPA sera but not against uncomplicated aspergilloma or normal sera. None of these low-molecular-weight components had any lectin-binding activity. Sera from patients with aspergilloma, however, frequently reacted with high-molecular-weight components of A. fumigatus. The majority of these high-molecular-weight antigenic components demonstrated concanavalin A-binding activity. The low-molecular-weight bands were discernible in Western blots with sera from all ABPA patients irrespective of disease activities, such as relapse, flare, or treatment. Antibodies detected by methods such as immunodiffusion or enzyme-linked immunosorbent assays demonstrated total antibody responses to most or all antigenic components, while Western blots demonstrated the reactivities of the individual components with the specific antibodies. Western blot analysis thus provided more information for immunodiagnosis of ABPA than other methods, especially when only crude antigens were available.     

Prolonged elevation of plasma argatroban in a cardiac transplant patient with a suspected history of heparin‐induced thrombocytopenia with thrombosis

BACKGROUND: Direct thrombin inhibitors (DTIs) provide an alternative method of anticoagulation for patients with a history of heparin‐induced thrombocytopenia (HIT) or HIT with thrombosis (HITT) undergoing cardiopulmonary bypass (CPB). In the following report, a 65‐year‐old critically ill patient with a suspected history of HITT was administered argatroban for anticoagulation on bypass during heart transplantation. The patient required massive transfusion support (55 units of red blood cells, 42 units of fresh‐frozen plasma, 40 units of cryoprecipitate, 40 units of platelets, and three doses of recombinant Factor VIIa) for severe intraoperative and postoperative bleeding. STUDY DESIGN AND METHODS: Plasma samples from before and after CPB were analyzed postoperatively for argatroban concentration using a modified ecarin clotting time (ECT) assay. RESULTS: Unexpectedly high concentrations of argatroban were measured in these samples (range, 0‐32 µg/mL), and a prolonged plasma argatroban half life (t_1/2) of 514 minutes was observed (published elimination t_1/2 is 39‐51 minutes [≤181 minutes with hepatic impairment]). CONCLUSIONS: Correlation of plasma argatroban concentration versus the patient's coagulation variables and clinical course suggest that prolonged elevated levels of plasma argatroban may have contributed to the patient's extended coagulopathy. Because DTIs do not have reversal agents, surgical teams and transfusion services should remain aware of the possibility of massive transfusion events during anticoagulation with these agents. This is the first report to measure plasma argatroban concentration in the context of CPB and extended coagulopathy.     

Dihydrofolate reductase as a target for chemotherapy in parasites

Opportunistic infections are known to cause morbidity and mortality in immunocompromised individuals. In addition, serious infections due to several parasites are also known to affect the quality and duration of life in normal individuals. The importance of dihydrofolate reductase (DHFR) in parasitic chemotherapy arises from its function in DNA biosynthesis and cell replication. DHFR catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), an essential cofactor in the biosynthesis of thymidylate monophosphate (dTMP). Inhibition of DHFR leads to a deficiency of dTMP since DHF cannot be recycled, and thus causes inhibition of cell growth. Methotrexate (MTX) and aminopterin (AMT) were among the first known classical inhibitors of DHFR. Trimethoprim (TMP) and pyrimethamine (PYR) are among the first known non classical inhibitors of DHFR. TMP and PYR are selective but weak inhibitors of DHFR from several parasitic organisms and coadministration of sulfonamides is required to provide synergistic effects for clinical utility. Unfortunately, the side effects associated with sulfa drugs in this combination often result in cessation of therapy. Trimetrexate (TMQ) and piritrexim (PTX) are two potent non classical inhibitors, neither of which exhibit selectivity for pathogen DHFR and must be used with host rescue. However, the current combination therapy suffers from high cost, in addition, several mutations have been reported in the active site of parasitic DHFR rendering the infections refractive to known DHFR inhibitors. The selectivity of TMP is a hallmark in the development of DHFR inhibitors and several efforts have been made to combine the potency of PTX and TMQ with the selectivity of TMP. Thus the structural requirements for DHFR inhibition are of critical importance in the design of antifolates for parasitic chemotherapy. Structural requirements for inhibition have been studied extensively and novel agents that exploit the differences in the active site of human and parasitic DHFR have been proposed. This review discusses the synthesis and structural requirements for selective DHFR inhibition and their relevance to parasitic chemotherapy, since 1995.     

Availability and activity of preservatives in emulsified systems.

Availability and activity of methyl-p-hydroxybenzoate, phenoxyethanol and chlorocresol in emulsions were studied by equilibrium dialysis and microbiological methods. Equation proposed by Bean and coworkers for calculating the availability of free preservatives in the aqueous phase of simple emulsion was also found to be applicable to modern emulsions composed of more than one oil and surfactant. The antimicrobial activity varied with the type of preservative and microorganism. Among the four test organisms, Pseudomonas aeruginosa and Staphylococcus aureus were more resistant to the various preservatives than Candida albicans and Aspergillus niger. The concentration of free preservative in the aqueous phase of the emulsion required to reduce the microbial population to the level specified in BP was found to be less than the concentration needed in distilled water indicating that the antimicrobial activity of the preservatives in emulsions is not entirely dependent on the concentration of their free form in the aqueous phase of emulsions.