Biomedical subjects
Publications and source records attributed to B J Luft.
Toxoplasmosis remains an important complication of AIDS. Recent advances in both diagnosis and treatment have decreased the immediate mortality. New innovations in prophylaxis will ultimately decrease incidence of this disease.
The chromosomal genes fla and p93 and the ospA gene from a linear plasmid were sequenced from up to 15 isolates of Borrelia burgdorferi, which causes Lyme borreliosis in man. Comparison of the gene trees provides no evidence for genetic exchange between chromosomal genes, suggesting B. burgdorferi is strictly clonal. Comparison of the chromosomal gene trees with that of the plasmid-encoded ospA reveals that plasmid transfer between clones is rare. Evidence for intragenic recombination was found in only a single ospA allele. The analysis reveals three common clones and a number of rare clones that are so highly divergent that vaccines developed against one are unlikely to provide immunity to organisms from others. Consequently, an understanding of the geographic and genetic variability of B. burgdorferi will prove essential for the development of effective vaccines and programs for control. While the major clones might be regarded as different species, the clonal population structure, the geographic localization, and the widespread incidence of Lyme disease suggest that B. burgdorferi should remain the name for the entire array of organisms.
Outer surface protein A (OspA) is a major antigen of Borrelia burgdorferi, the etiological agent of Lyme disease. A recombinant form of OspA (OspA-257) from B. burgdorferi, strain B31, contains 257 amino acids and a single tryptophan residue at position 216 (Trp-216). Mapping studies indicate that Trp-216 is involved in the epitope for the agglutinating monoclonal antibody 105.5. However, the fluorescence emission maximum of the native protein is 330 nm, indicating that Trp-216 is not solvent-exposed. Primary structure analysis suggests an alpha-helical conformation for residues approx. 204-217, which, if located on the protein surface, would allow Trp-216 to be buried, while leaving hydrophilic residues on the opposite side of the helix exposed. This helix would place Lys-212 within approx. 6 A of Trp-216; the presence of such a positively-charged residue can, in principle, be ascertained from fluorescence quenching studies. Stern-Volmer plots confirm that Trp-216 is indeed buried in the native protein, but is readily accessible to the small polar quencher, acrylamide. Furthermore, the dominant component of the fluorescence emission shows only weak dynamic quenching by the positively-charged quencher, Cs+, while the minor component undergoes static quenching by I-, indicating the proximity of a positively-charged residue. These data are consistent with the existence of an alpha-helix from residues 204-217 in the predicted orientation at the protein surface, hence indicating the structure of the antigenic determinant.
BACKGROUND: In patients with the acquired immunodeficiency syndrome (AIDS), toxoplasmic encephalitis is usually a presumptive diagnosis based on the clinical manifestations, a positive antitoxoplasma-antibody titer, and characteristic neuroradiologic abnormalities. A response to specific therapy helps to confirm the diagnosis, but it is unclear how rapid the response should be. We studied the course of patients treated for acute toxoplasmic encephalitis and evaluated objective clinical criteria for this empirical diagnosis. METHODS: A quantifiable neurologic assessment was used prospectively to evaluate the clinical outcome of patients with AIDS and toxoplasmic encephalitis who were treated with oral clindamycin (600 mg four times a day) and pyrimethamine (75 mg every day) for six weeks. RESULTS: Thirty-five of 49 patients (71 percent) responded to therapy, and 30 of these (86 percent) had improvement by day 7. Thirty-two of those with a response (91 percent) improved with respect to at least half of their base-line abnormalities by day 14. Improvement in neurologic abnormalities within 7 to 14 days after the start of therapy was strongly associated with the neurologic response at 6 weeks. The four patients in whom treatment failed and the two patients with lymphoma had progressing neurologic abnormalities or new abnormalities during the first 12 days of therapy. Nonlocalizing abnormalities (headache and seizure) improved regardless of the clinical outcome. CONCLUSIONS: Oral clindamycin and pyrimethamine are an effective treatment for toxoplasmic encephalitis. Patients who have early neurologic deterioration despite treatment or who do not improve neurologically after 10 to 14 days of appropriate antitoxoplasma therapy should be considered candidates for brain biopsy.
PURPOSE: To examine the cost-effectiveness of empirical, parenteral antibiotic treatment of patients with chronic fatigue and myalgia and a positive serologic result for Lyme disease who lack classic manifestations. DATA SOURCES: Peer-reviewed journals, opinion of experts in the field, and published epidemiologic reports. STUDY SELECTION: Consensus by authors on articles that indicated methods for patient selection; on criteria used for diagnosis; on immunologic methods used for classifying patients; on the dose and duration of therapy; and on criteria by which responses to therapy were ascertained. DATA EXTRACTION: In a cost-effectiveness model, the costs and benefits of empirical parenteral therapy for patients seropositive for Lyme disease were compared with a strategy in which only patients having classical symptoms of Lyme disease were treated. DATA SYNTHESIS: In areas endemic for Lyme disease, the incidence of false-positive serologic results in patients with nonspecific myalgia or fatigue exceeds by four to one the incidence of true-positive results in patients with nonclassical infections. Treatment of the former group of patients costs $86,221 for each true-positive patient treated. The empirical strategy causes 29 cases of drug toxicity for every case in the more conservative strategy. If patients were willing to pay $3485 to eliminate anxiety about not treating possible true Lyme disease, the empirical strategy would break even. CONCLUSION: For most patients with a positive Lyme antibody titer whose only symptoms are nonspecific myalgia or fatigue the risks and costs of empirical parenteral antibiotic therapy exceed the benefits. Only when the value of patient anxiety about leaving a positive test untreated exceeds the cost of such therapy is the empirical treatment cost-effective.
The p41 flagellin of Borrelia burgdorferi is the most common antigen recognized by serum of patients with Lyme borreliosis. This antigen shares amino acid homology, particularly in the amino and carboxy termini, with periflagellar antigens found in other microorganisms including Treponema pallidum. We cloned and expressed the p41 open reading frame in Escherichia coli and expressed it both as TrpE fusion and full-length unfused proteins. Also, we generated deletion constructs of various portions of the gene. Sera from patients with late Lyme borreliosis and secondary syphilis were used to identify the recombinant proteins by immunoblot analysis. Sera from 26 patients with Lyme borreliosis, 20 with secondary syphilis and 10 controls were used to identify cross-reactive domains of the B. burgdorferi flagellin. The variable region (amino acids 131-234) of the protein was recognized by 59% (15/26) of patients with late Lyme borreliosis compared to 30% (6/20) of patients with secondary syphilis and no (0/10) control patients. It appears that cross-reactive epitopes between B. burgdorferi and T. pallidum extend to the variable region of the flagellin.
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The flagellin gene from the P/Gau strain of Borrelia burgdorferi was cloned and sequenced. The translated P/Gau flagellin protein differed from the flagellin of the B31 strain at 13 of 336 amino acids. This includes seven differences between amino acids 190-234, an immunodominant and specific region for B. burgdorferi. The entire flagellin molecule, as well as peptides of the internal portion of the protein which is more specific for B. burgdorferi, has been expressed in Escherichia coli using a pET7HIS.2 expression system. These peptides may be of great value for the development of sensitive and specific recombinant-based serological assays.
OBJECTIVE: To determine central nervous system (CNS) involvement in acutely disseminated Borrelia burgdorferi infection by measurement of borrelia-specific DNA using the polymerase chain-reaction (PCR) assay and to compare the results of this with standard serological tests. DESIGN: Prospective study with laboratory investigators blinded to clinical data. SETTING: Multicenter office practice with a central reference laboratory. PATIENTS: Cerebrospinal fluid (CSF) was collected from 12 patients with acute disseminated Lyme borreliosis with less than 2 weeks of active disease. The normal control specimens came from 16 patients whose CSF samples had been sent to the clinical laboratory for tests unrelated to the present study. MAIN OUTCOME MEASURES: Clinical evidence of disease and laboratory abnormalities. RESULTS: Eight of the 12 patients (four of six with multiple areas of erythema migrans and four of six with cranial neuritis without erythema migrans) had B burgdorferi-specific DNA in their CSF. Among the 12 patients studied, nine had acute cranial neuritis and six had multiple erythema migrans lesions. Just four of the eight who were found to have spirochetal DNA in their CSF had complaints suggestive of CNS infection. In three of the PCR-positive CSF samples, no other abnormalities were noted. None of 16 samples from controls were positive in the PCR assay. CONCLUSION: B burgdorferi can invade the CNS early in the course of infection. Careful consideration should be given to choosing antibiotics that achieve adequate CSF levels in patients with disseminated infection.
Involvement of the central nervous system (CNS) is common in patients with advanced disease due to human immunodeficiency virus (HIV). Symptoms range from lethargy and apathy to coma, incoordination and ataxia to hemiparesis, loss of memory to severe dementia, and focal to major motor seizures. Involvement may be closely associated with HIV infection per se, as in the AIDS dementia complex, but is frequently caused by opportunistic pathogens such as Toxoplasma gondii and Cryptococcus neoformans or malignancies such as primary lymphoma of the CNS. The clinical presentations of attendant and direct CNS involvement are remarkably non-specific and overlapping, yet a correct diagnosis is critical to successful intervention. Toxoplasmic encephalitis is one of the most common and most treatable causes of AIDS-associated pathology of the CNS. A great deal has been learned in the last 10 years about its unique presentation in the HIV-infected patient with advanced disease. Drs. Benjamin J. Luft of the State University of New York at Stony Brook and Jack S. Remington of the Stanford University School of Medicine and Palo Alto Medical Foundation's Research Institute have studied T. gondii for many years and are two of the leading experts in the field. This commentary comprises an update of their initial review (J Infect Dis 1988;157:1-6) and a presentation of the current approaches to diagnosing and managing toxoplasmic encephalitis in HIV-infected patients.
Using immunoblots, we identified proteins of Borrelia burgdorferi recognized by sera from 62 patients with either acute or chronic Lyme disease. In all groups studied, the 41-kDa flagellar protein and a relatively minor 93-kDa protein (p93) were the most commonly recognized antigens in patients with acute and chronic disease due to B. burgdorferi. A murine monoclonal antibody (MAb 181.1) was developed against p93, and the antigen was detected by immunoblot analysis in four European and American strains of B. burgdorferi. On two-dimensional gel electrophoresis, p93 had an apparent pI of 6.8. Immunoelectronmicroscopy with MAb 181.1 demonstrated that p93 is located within the protoplasmic cylinder compartment of the organism. The gene encoding p93 was retrieved from a phage expression library. The derived amino acid sequence of p93 confirmed chemical characterization of the antigen, including its amino-terminal peptide sequence. The derived amino acid sequence predicted it to be predominantly alpha helical. A prominent antigenic domain located at the carboxy portion of the protein was recognized by human and rabbit polyclonal antisera and human (MAb D4) and mouse (MAb 181.1) MAbs.
The gene encoding a Borrelia burgdorferi DnaJ homolog, located immediately 3' of the hsp70 gene, was characterized. Although there is a single copy of the dnaJ gene on the spirochetal chromosome, two distinct dnaJ transcripts are detected in B. burgdorferi RNA. RNA blot analysis indicates that the dnaJ gene can be transcribed alone or as part of a larger transcript containing the hsp70 homolog.
The gene encoding an immunoreactive Borrelia burgdorferi HSP70 homolog was isolated and characterized. The predicted amino acid sequence of this spirochetal protein confirms that this gene encodes a member of the HSP70 family of proteins. Although there appears to be a single copy of this gene on the spirochetal chromosome, two distinct transcripts hybridizing to the hsp70 probe are detected in RNA isolated from B. burgdorferi. The amount of spirochetal HSP70 RNA transcripts is shown to be thermally regulated. Antibodies in the serum of three Lyme arthritis patients and cloned T-cell lines isolated from one patient with Lyme arthritis recognize the expressed recombinant HSP70, indicating that it is an immunologically important spirochetal antigen. Antibodies in a rabbit antiserum, as well as antibodies in the serum of two of three Lyme arthritis patients examined, bound to expressed truncated recombinant HSP70s with 250 amino acids deleted from either the amino or carboxy terminus of the protein. However, antibodies in the serum of three Lyme arthritis patients, which were reactive with spirochetal HSP70, did not cross-react with human HSP70 proteins.
Purified flagellar protein (p41) of Borrelia burgdorferi (strain B31) was subjected to chemical cleavage with hydroxylamine or proteolysis with V8 protease, endoproteinase Asp-N, or alpha-chymotrypsin. The resulting polypeptides were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their positions in the published DNA sequence of the p41 protein were determined by amino-terminal sequencing and amino acid analysis. Epitope specificities of antibody binding by a monoclonal antibody raised by immunization of mice with purified flagella and pooled sera from patients with multiple erythema migrans, late Lyme borreliosis, or secondary syphilis were analyzed by Western blots (immunoblots) of peptides transferred to Immobilon polyvinylidene difluoride filters. The major epitope binding one murine monoclonal antibody (158) was localized to a carboxy-terminal domain that includes residues 300 to 336. The dominant epitopes binding human polyclonal antibodies are in the central portion of the molecule (residues 182 to 218) that is not conserved compared with other bacterial flagellins. Additional reactive epitopes were identified in the amino-terminal domain of the protein. Sera from patients with syphilis bound strongly to the amino-terminal conserved domain, providing a structural basis for cross-reactivity seen in standard enzyme-linked immunosorbent assays, but not to the central part of the molecule. Specific and cross-reactive antigenic determinants need to be considered in the design of improved immunodiagnostics for spirochetal diseases.
Central nervous system toxoplasmosis is a major opportunistic infection in patients with acquired immunodeficiency syndrome. The standard therapy for this infection is pyrimethamine (PYR) and sulfonamides. To assess in vivo if PYR alone could adequately treat toxoplasmosis, a murine model of acute toxoplasmosis was used. The CD1 strain of mice was infected intraperitoneally with 10(4) parasites of the RH strain of Toxoplasma gondii. Pyrimethamine was administered in mouse chow at concentrations of 0, 0.03125, 0.0625, 0.125, 0.25, or 1.0 mg of PYR/g of food, which provides the following daily PYR dosages: 0, 6.25, 12.5, 25, 50, and 200 mg/kg/day. No sulfonamides were administered. Serum PYR levels proved more accurate than mg of PYR/g of food in predicting survival. Mice with serum PYR levels greater than or equal to 500 ng/ml (2 microM) survived and had no parasites present on peritoneal lavage. Mice with serum PYR levels less than 100 ng/ml (0.4 microM) had a 100% mortality rate and the average parasite count was 3 x 10(7) organisms in the lavage fluid. At a PYR level of 370 ng/ml, six of 11 mice survived and the lavage fluid contained 2.5 x 10(5) organisms. Previously, using 3H-uracil in an in vitro assay, PYR at a concentration of 500 ng/ml was shown to be as effective in inhibiting Toxoplasma growth as the combination of PYR (100 ng/ml) and sulfonamides 25 micrograms/ml). These data suggest the potential usefulness of PYR for monotherapy of toxoplasmosis and are consistent with previously described in vitro assays.
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Lyme borreliosis is an infectious disease caused by the tick-borne spirochete Borrelia burgdorferi, which carries the potential for chronic infection. Ag on the etiologic Borrelia are currently being defined structurally and their ability to elicit immune responses delineated. EBV can be used to immortalize human B. burgdorferi-specific B cells from infected donors and generate antibodies against antigenic epitopes encountered in natural infection. A human mAb secreting EBV-transformed B cell line, D7, has been developed that is specific for a 93-kDa B. burgdorferi protein and has been used to characterize this potentially important Ag. D7 produces an IgG3 antibody that detects the 93-kDa Ag as well as smaller fragments at 46 kDa and lower molecular mass. The antibody detects similar epitopes on all B. burgdorferi isolates tested and on a Borrelia hermsii protein with molecular mass greater than 100 kDa but binds poorly to Treponema species. In contrast, polyclonal sera from Lyme disease patients show little binding to the homologous Ag in B. hermsii. Structurally, the 93-kDa protein is associated with the flagellum and may be firmly anchored in the protoplasmic cylinder. It is not solubilized by nonionic detergent treatment of the whole Borrelia. Antibodies against a comparable m.w. protein are present in sera from patients with both early and late infection. Thus, antibodies against this Ag are a sensitive and specific marker of Borrelia infection. This Ag is likely of structural importance and may represent a target of host defenses.
The 66- to 73-kDa proteins of Borrelia burgdorferi are dominant immunogens and expressed in all strains of B. burgdorferi. The humoral response to these Ag occurs relatively early during the course of infection. Two-dimensional Western blot analysis of this group of Ag revealed them to consist of a tetrad of proteins with apparent molecular mass of 66, 68, 71, and 73 kDa. Furthermore, in this study we demonstrate the 66-kDa protein to be a potent inducer of lymphoproliferation in the patient immune to B. burgdorferi. Monospecific polyclonal antibodies and mAb demonstrate that each of these proteins was immunologically distinct. However, direct amino acid sequence of the 66- and 68-kDa Ag was almost identical and had a high level of sequence similarity to the GroEL heat-shock protein (Hsp60) of Escherichia coli and the 60-kDa immunodominant protein of Treponema pallidum. The amino terminal sequence of the 71- and 73-kDa proteins of B. burgdorferi was almost identical and these proteins had remarkable sequence similarity to the DnaK heat-shock protein of E. coli (Hsp70). It appears likely, therefore, that proteins related to the heat-shock family are potent immunogens of B. burgdorferi.