T-cell epitope mapping of the Borrelia garinii outer surface protein A in lyme neuroborreliosis.

We studied the T-cell reactivity to overlapping peptides of B. garinii OspA, in order to locate possible immunodominant T-cell epitopes in neuroborreliosis. Cells from cerebrospinal fluid (CSF) and blood from 39 patients with neuroborreliosis and 31 controls were stimulated with 31 overlapping peptides, and interferon-gamma secreting cells were detected by ELISPOT. The peptides OspA(17-36), OspA(49-68), OspA(105-124), OspA(137-156), OspA(193-212) and OspA(233-252) showed the highest frequency of positive responses, being positive in CSF from 38% to 50% of patients with neuroborreliosis. These peptides also elicited higher responses in CSF compared with controls (P = 0.004). CSF cells more often showed positive responses to these peptides than blood cells (P = 0.001), in line with a compartmentalization to the central nervous system. Thus, a set of potential T-cell epitopes were identified in CSF cells from patients with neuroborreliosis. Further studies may reveal whether these epitopes can be used diagnostically and studies involving HLA interactions may show their possible pathogenetic importance.

Dependence of the in vitro antiproliferative activity of recombinant human gamma-interferon on the concentration of tryptophan in culture media.

In addition to its antiviral and antibacterial activities, recombinant human gamma-interferon (rHuIFN-gamma) can exert an antiproliferative effect on human cell lines. The mechanisms involved in this antiproliferative activity are poorly understood, but it is known that IFN-gamma can induce indoleamine 2,3-dioxygenase, which enhances tryptophan metabolism and thus depletes the cellular pool of this amino acid. In the present study we have examined the effect of different tryptophan concentrations on the antiproliferative activity of rHuIFN-gamma on four human tumor cell lines, HeLa 229, HEp-2, A549, and T24. Cells were grown in the presence of rHuIFN-gamma (0.01 to 100 ng/ml) and/or tryptophan (10 to 400 micrograms/ml) for 7 days at which time they were counted. rHuIFN-gamma (4 ng/ml) inhibited the growth of A549 and T24 cells by 50%. Hep-2 and HeLa 229 cells were more sensitive to the rHuIFN-gamma induced antiproliferative effects, requiring only 0.4 ng/ml for a 50% inhibition. Addition of tryptophan to the media at concentrations from 10 to 100 micrograms/ml resulted in a significant blockage of the antiproliferative activity of rHuIFN-gamma. For example, when 50 micrograms/ml of tryptophan were added to the media, 10 times more rHuIFN-gamma (4 ng/ml) was needed to inhibit HeLa 229 cells by 50% of the control. The A549 was the most sensitive cell line to the modulatory activity of the tryptophan. Addition of 10 micrograms/ml of tryptophan changed the amount of rHuIFN-gamma needed to produce a 50% inhibition from 4 ng/ml to 100 ng/ml. In summary, in the four human tumor cell lines tested, the antiproliferative activity of rHuIFN-gamma could be modulated by the concentration of tryptophan in the media.

The effect of orientation within a chimeric peptide on the immunogenicity of Chlamydia trachomatis epitopes.

Peptides representing the Chlamydia trachomatis major outer membrane protein variable domains (VD) 1 and 4 of serovars C and E, respectively, have been shown to elicit a neutralizing antibody response in mice. To assess whether the position within a chimeric peptide influences the immunogenicity of the epitopes, two constructs, VD 1-4 and VD 4-1, were made in which the position of the VD relative to the amino and carboxy terminals were rotated. C57BL/10 mice were immunized with 100 micrograms of peptide in complete Freund's adjuvant (FA) on day 0, followed by an immunization with peptide (100 micrograms) in complete FA on day 14. By day 21 the immunodominant epitope in both chimeras as measured by ELISA was the one located at the carboxy terminus. A pepscan of the VD 1-4 antisera revealed a main peak in VD 4 which had been previously identified by neutralizing MAbs. The VD 4-1 antisera gave a peak in the VD 1 region which did not correspond to regions previously mapped with neutralizing MAbs. The VD 1-4 antisera but not the VD 4-1 antisera was able to neutralize in vitro serovar E. In summary, the position of these chlamydial epitopes within a chimeric peptide greatly influenced the resulting immune response.

Nucleotide sequence of DNA encoding the major outer membrane protein of Chlamydia trachomatis serovar L3.

DNA encoding the major outer membrane protein of Chlamydia trachomatis serovar L3 was sequenced following amplification by the polymerase chain reaction. A comparison with the deduced amino acid (aa) sequence of the C. trachomatis serovar L2 showed that the L3 had three extra aa and 55 aa substitutions.

Sequence of the gene encoding the major outer membrane protein of the mouse pneumonitis biovar of Chlamydia trachomatis.

The gene encoding the major outer membrane protein of the Chlamydia trachomatis mouse pneumonitis biovar was sequenced and the amino acid sequence deduced. The primary structure of this protein is similar to that of the lymphogranuloma venereum and trachoma biovars in that it consists of four variable domains interspersed with five constant domains. This protein may be an ideal candidate for a vaccine in chlamydia-infected mouse experimental models.

Synthesis and biological evaluation of 5'-sulfamoylated purinyl carbocyclic nucleosides.

The first series of 5'-sulfamoylated carbocyclic purinyl nucleosides was synthesized and tested for antitumor and antibacterial activities. The target compounds were formed by reacting the 2',3'-acetonide-protected carbocyclic nucleosides with sulfamoyl chloride, followed by deprotection. The agents were tested for cytotoxic activity against P388 mouse leukemia cells. Two compounds, 5'-sulfamoyl carbocyclic adenosine (2) and 5-sulfamoyl-8-aza carbocyclic adenosine (6) exhibited IC50 values as low as 62 and 15 nM, respectively. These analogs inhibited protein biosynthesis and slowed down DNA and RNA biosyntheses in the P388 cells. None of the target molecules were as potent against Escherichia coli as they were against the tumor cells. Also, in cell-free systems, agents 2 and 6 were more effective inhibitors of protein synthesis in rabbit reticulocyte lysate than in E. coli. These new carbocyclic derivatives appear to be somewhat selective for eukaryotic over prokaryotic cells in affecting translation.

Alpha-spirocyclopentyl- and alpha-spirocyclopropyl-gamma-butyrolactones: conformationally constrained derivatives of anticonvulsant and convulsant alpha,alpha-disubstituted gamma-butyrolactones.

To further study the putative gamma-butyrolactone site of the GABAA/chloride channel complex, constrained derivatives of convulsant and anticonvulsant alpha,alpha-disubstituted gamma-butyrolactones (alpha-spirocyclopropyl- and alpha-spirocyclopentyl-gamma-butyrolactones) were synthesized and evaluated biologically. Most of the spirocyclopropyl agents were anticonvulsants when tested against pentylenetetrazole-induced seizures in mice. These agents effectively displaced 35[S]-tert-butylbicyclophosphorothionate (35[S]-TBPS), a ligand for the picrotoxin binding site of the GABAA/chloride channel, from rat neuronal membranes and affected the GABA-mediated current in hippocampal neurons. The monomethyl-substituted spirocyclopropyl agent with a methyl group cis to the carbonyl (15) potentiates GABA-induced current whereas the trans derivative (16) blocks the current. The only anticonvulsant in the spirocyclopentyl series was the unsubstituted spirocyclopentyl compound 2. All the other substituted spirocyclopentyl targets were inactive in vivo at the highest dose tested except for convulsant 9, which has a trans 2,5-dimethyl-substituted cyclopentyl ring. All the spirocyclopentyl derivatives displaced 35[S]-TBPS from rat neuronal membranes very effectively, and they also all potentiated GABA-induced chloride current except for convulsant 9 which blocked the current. From the data obtained in this investigation, it appears that when the volume occupied above and below the lactone ring is as large as that occupied by spirocyclopentyl agent 9, convulsant activity is observed. Groups with less volume in these areas either are inactive in the behavioral test or have anticonvulsant activity. When bound to the GABAA/chloride channel, the larger molecules may stabilize the closed state of the channel whereas the smaller molecules may stabilize the open state.

Detection of antibodies to Coccidioides immitis by enzyme immunoassay.

Two hundred twenty-six specimens (160 serum samples, 66 cerebrospinal fluid samples) were tested with the Meridian Premier Coccidioides enzyme immunoassay (MPC-EIA), and the results were compared with those of conventional serologic tests detecting complement-fixing (CF) and tube precipitin (TP) antibodies. Of the 73 positive specimens by CF, 72 were positive by MPC-EIA (72 IgG positive, 48 IgM positive) and 1 was indeterminate. Of the 132 specimens negative by CF, 126 were negative by MPC-EIA, 3 were IgM positive only, and 3 were indeterminate. Of the three patients with specimens positive for IgM only, CF demonstrated seroconversion within a month in one. All 21 specimens that displayed anticomplementary activity by CF were confirmed negative by agar gel immunodiffusion for CF antibodies (IDCF) and TP antibodies (IDTP); 20 of these serum samples were also negative by MPC-EIA, and 1 was indeterminate. After exclusion of the 25 specimens with anticomplementary or indeterminate activity, results of MPC-EIA were in agreement with those of CF in 99% of specimens. With CF as reference, MPC-EIA had a specificity of 98%, sensitivity of 100%, and positive and negative predictive values of 96% and 100%, respectively. Correlation coefficient values from linear regressions of log-2 CF titer vs MPC-EIA IgG index values were calculated at .290 for serum samples and .931 for cerebrospinal fluid.

The effect of media and temperature on the storage of Chlamydia trachomatis.

Four media, Eagle's minimal essential medium with 10% fetal bovine serum (MEM/FBS), tryptic soy broth (TSB), 2-SP, and 4-SP, were compared for their ability to maintain the viability of Chlamydia trachomatis at 4 degrees C, -20 degrees C, -70 degrees C, and -176 degrees C (liquid nitrogen) over a 1-week period. 2-SP maintained viability of C. trachomatis to the greatest extent for all of the time intervals and temperatures examined. Therefore, in an attempt to further stabilize the viability of C. trachomatis, 2-SP was supplemented with various concentrations of bovine serum albumin (BSA) or fetal bovine serum (FBS). For the times and temperatures examined, 2-SP supplemented with 20% or 40% FBS or 5% BSA preserved infectivity to a greater extent than unsupplemented 2-SP. In some supplemented media, up to 65% of the infectivity was preserved after one week of storage at -176 degrees C, whereas only 0-3% of infectivity remained when stored in unsupplemented media at -20 degrees C and 4 degrees C, respectively. Therefore, supplementation of 2-SP with FBS or BSA can prolong the survival of chlamydia, which is critical in the transportation and storage of clinical specimens. In addition, storage for prolonged periods of time should be at -70 degrees C or lower temperatures.

Identification of viridans streptococci by three commercial systems.

The API 20S (Analytab Products, Plainview, NY), the GPI card (Vitek Systems, St. Louis, MO) and the RapSTR system (Innovative Diagnostics, Atlanta, GA) were compared with conventional biochemicals for the identification of viridans streptococci. One hundred nine clinical isolates were tested that included the following species: intermedius (38) sanguis II (20), bovis (variant) (14), mitis (14), salivarius (11), sanguis I (6), constellatus (3), mutans (2), and uberis (1). With initial testing, a correct species call was made with 72% of the isolates with the GPI card, 62% with the RapSTR, and 50% with the API 20S. Identifications of viridans streptococci group or those that needed additional biochemicals for species identification occurred with 28% of isolates with the API 20S, 8% with the RapSTR, and 9% with the GPI card. Incorrect identifications occurred with 6% of the isolates tested by the GPI card, 20% with the API 20S, and 30% with the RapSTR. Most discrepancies with the RapSTR were with 66% of the intermedius isolates, whereas most, 55%, of misidentifications with the API 20S were with sanguis II isolates. No identifications were made with 2% and 13% of isolates with the API 20S and GPI, respectively.

Evaluation of four anti-microbic susceptibility testing systems for gram-negative bacilli.

A total of 200 clinical isolates were assayed by five anti-microbic susceptibility testing systems. Two frozen minimal inhibitory concentration (MIC) systems (MicroScan and Pasco), an automated MIC system (AMS, Vitek Systems), and the standard disk diffusion were compared with a reference broth dilution method. Organisms tested included 100 resistant clinical stock strains and 100 fresh random clinical isolates. Overall, there were 1,600 anti-microbic-organism combinations analyzed. The Pasco and MicroScan systems had no major discrepancies, the AMS system had seven, and the disk diffusion two. The number of very major discrepancies were as follows: AMS, 11; disk diffusion, 9; MicroScan, 5; Pasco, 2. Of the total 36 major or very major discrepancies in the study, 33% (12 of 36) were with an aminoglycoside and 44% (16 of 36) occurred with a second-generation cephalosporin, of which 10 of 16 were with cefamandole. Overall, there was a greater than 98.8% essential agreement with all systems compared with the reference method.

Microorganisms in semen used for artificial insemination.

The effect of freezing semen in a cryopreservative media consisting of egg yolk glycerol with or without erythromycin was tested for its effect on the viability of microorganisms present in donor semen and on sexually transmitted pathogens seeded into semen. All donor semen contained two or three species of microorganisms that could be considered skin flora. Five of ten donor semen specimens contained Ureaplasma urealyticum that was not affected by either freezing or antimicrobial treatment. Some strains of Neisseria gonorrhoeae, when seeded into semen, survived all conditions except freezing in egg yolk glycerol containing erythromycin. Chlamydia trachomatis was erradicated when erythromycin was present in the cryopreservative. There was no detectable effect of any treatment tested on the survival of herpes simplex virus.

Improvement of positive blood culture detection by agitation.

To evaluate the advantages of agitation in reducing the detection time and increasing the recovery rate of positive blood cultures, 1,000 three-bottle sets of tryptic soy broth on adult inpatients were analyzed. Two bottles were transiently vented, one of which was agitated (250 rpm) for 7-19 hr at 35 degrees C. The other vented bottle and the anaerobic bottle were incubated stationary at 35 degrees C. Smears and subcultures were performed 7-19 hr after collection on both agitated and nonagitated vented bottles. Subcultures were done on all bottles at 72 hr and smears were performed on the anaerobic bottle. There were 137 of 1000 (13.7%) positive cultures from 90 patients. The agitated bottle detected 112 of 137 (81.8%) positive cultures, was the first or only means of detection in 57 of 137 cultures (41.6%), and was the only positive bottle in 30 of 137 (21.9%) cultures. The nonagitated vented bottle detected 89 of 137 (65.0%) of positive cultures and was the only means of detection in 13 of 137 (9.5%), but was never the first means of detection. The anaerobic bottle detected 76 of 137 (55.5%) of positive cultures, was the first or only means of detection in 11 of 137 (8.0%), and was the first means of detection in one of 137 (0.7%) cultures. When both the agitated and nonagitated bottle were positive, the agitated bottle was positive on the average 35 hr earlier. We conclude that agitation of the vented bottle in a conventional blood culture system significantly decreases the detection time of positive blood cultures and increases the number of positive blood cultures detected.

Evaluation of a new herpes simplex virus typing reagent for tissue culture confirmation.

Monoclonal antibody typing reagents for herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) from California Integrated Diagnostics, Inc. (Berkeley, CA) were compared to two other commercially available HSV monoclonal antibody typing reagents. Of the 105 specimens tested, of which 81 were positive for HSV, there was 100% agreement with all three typing reagents.

Rotavirus gastroenteritis in southern California.

The epidemiology of rotavirus infections in Southern California was analyzed over a three year period, from January 1, 1981 through December 31, 1983. Data was available from patients seen at the University of California Irvine Medical Center (UCIMC), in addition to referral testing provided to the community in Orange County. Over the 3 yr period the laboratory performed 1172 rotavirus assays. Out of these, 345 were positive for an overall positive rate of 29.4%. The 643 stool specimens from UCIMC corresponded to 508 patients, of which 31.1% (158/508) were positive for rotavirus. The majority of patients with a positive rotavirus test were under 1 yr of age (117/158), with only ten cases found in the 2-15 yr old group. The distribution of the positive rotavirus tests was similar for the female and male population. Approximately 70% of the positive results occur during October through December, with the month of November having the highest incidence. The distribution of positive rotavirus tests did not appear to correlate with either the coldest or the driest month of the year in Southern California.

Evaluation of five cell types for the isolation of herpes simplex virus.

Five cells were evaluated in a comparative analysis for sensitivity, specificity, and rapidity in detecting the presence of herpes simplex virus HSV-1 and HSV-2. Included in this study were human embryonic kidney (HEK), rabbit kidney (RK), MRC-5, mink lung (ML), and Microtus agrestis (UMMA). A total of 274 specimens from genital, throat, skin, or other sources that were submitted for HSV isolation were used in the study. The sensitivity of the different cells was assessed by the total number of positive cultures detected by all the cells under evaluation. At 48 hr, HEK and RK detected 80% of the positives, ML detected 79%, MRC-5 detected 73%, and UMMA detected 60%. All cells tested were satisfactory; however, the choice of which cell to use for isolation of HSV depends upon the needs of the specific laboratory.

Comparison of the ProSpecT and Color Vue enzyme-linked immunoassays for the detection of Cryptosporidium in stool specimens.

Two enzyme-linked immunosorbent assays, the ProSpecT (Al-exon, Sunnyvale, CA, USA) and the Color Vue (Seradyn, Indianapolis, IN, USA) were compared for their ability to detect Cryptosporidium in 236 formalin-fixed stool specimens using the Merifluor C/G (Meridian, Cincinnati, OH, USA) stain as the reference method. The initial sensitivities of the ProSpecT and the Color Vue were 96.0% and 76.0%, which upon repeat testing of all discrepancies remained at 96.0% for the ProSpecT and decreased to 72.0% for the Color Vue. There were 25 (11%) specimens positive by the reference method. Initially, there were five false positive specimens by the ProSpecT, only one of which remained positive on retesting. The specificity of the Color Vue was 100% for the initial and repeated results, whereas the ProspecT had an initial specificity of 97.6% that increased to 99.5% upon repeat testing. The enzyme-linked immunosorbent assays offered the advantages of objectivity, batch testing, and, in the case of the ProSpecT, an acceptable sensitivity.

Evaluation of the Cellmatics and direct monoclonal fluorescence antibody staining for detection of genital chlamydial infections.

Three methods for Chlamydia trachomatis detection were compared: the Cellmatics (Difco Laboratories, Detroit, MI), a commercially available tissue culture system which contains a rat cell line; a direct fluorescent Chlamydia antibody (DFA) (Microtrak, Syva Corp., Palo Alto, California) stain; and a standard tissue culture isolation method employing McCoy cells. Of the 121 specimens in the study, 20 were positive by the standard cell culture. All 20 of these specimens were also positive by the Cellmatics but only 10 were positive by DFA.

Evaluation of a shell vial centrifugation method for the detection of herpes simplex virus.

A MRC-5 shell vial method was compared to a MRC-5 conventional tube cell culture method in 410 specimens, 88 of which were positive for herpes simplex virus. The shell vial had a sensitivity of 92% and a specificity of 99% when compared to conventional cell culture.

Factors influencing the induction of infertility in a mouse model of Chlamydia trachomatis ascending genital tract infection.

In women, infections due to Chlamydia trachomatis frequently result in long-term sequelae including chronic abdominal pain, ectopic pregnancy and infertility. In an attempt to characterise the pathogenesis of the infection, female C3H (H-2k) mice were inoculated intravaginally with different doses of C. trachomatis and then mated with proven male breeder mice. The inoculated mice developed a broad spectrum of clinical manifestations ranging from infertility to asymptomatic shedding. The dose inducing infertility in 50% of the mice was c. 10(5) inclusion-forming units of C. trachomatis. In another group of mice sampled at intervals after intravaginal inoculation, C. trachomatis was recovered from the upper genital tract starting at 24 h after infection. A higher percentage of animals infected during the luteal phase of the oestrous cycle had positive cultures from the middle and upper genital tract than when mice were inoculated during the follicular phase. These results indicate that rapid therapeutic intervention is required to avoid the sequelae resulting from C. trachomatis genital infection, and suggest that hormonal factors play a role in the pathogenesis of the disease.