High-resolution mapping of serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis.

The principal surface protein antigen of Chlamydia trachomatis is the major outer membrane protein (MOMP). The MOMP is antigenically complex. Among the 15 serovars of C. trachomatis, mAbs define serovar-, subspecies-, and species-specific determinants on MOMP. The molecular basis of the antigenic diversity of these proteins is reflected in amino acid variable sequence domains. We have mapped the dominant topographic antigenic determinants of MOMP that are defined by mAbs. Using recombinant DNA approaches we have identified the linear distribution of two antigenic domains. One domain contains a serovar-specific determinant and the other contains subspecies- and species-specific determinants. These antigenic domains correspond to two amino acid sequence variable domains. Synthetic peptides were immunogenic and these resolved the serovar-specific determinant within a 14-amino acid peptide. The subspecies- and species-specific determinants were overlapping within a 16-amino acid peptide.

Rabbit model of Lyme borreliosis: erythema migrans, infection-derived immunity, and identification of Borrelia burgdorferi proteins associated with virulence and protective immunity.

Erythema migrans (EM), persistent skin infection, and visceral dissemination can be induced reproducibly in the adult male New Zealand White rabbit by intradermal injection of as few as 10(3) Borrelia burgdorferi. EM was found to persist for 7 +/- 3 d. Skin culture positivity (infection) cleared within a mean of 6.7 +/- 1.4 wk after infection and similarly visceral infection was not demonstrated after 8 wk; infection-derived immunity to intradermal challenge was evident 5 mo after initial infection. The extent of the protection against EM and dermal infection induced by untreated infection was directly related to the extent of prior in vitro passage of the B31 strain. Initial infection with as few as 4 x 10(3) B31 passage 4 induced complete protection against EM and skin infection upon subsequent challenge with 4 x 10(7) B31, passage 4. Initial infection with B31 passage 27 led to partial protection against EM along with complete protection against skin infection. Initial infection with passage 47 led to partial protection against EM, but conferred no protection against skin infection. Using serum from rabbits fully immune to reinfection, we defined a set of B. burgdorferi proteins present in virulent B31, but absent in the avirulent American Type Culture Collection B31 strain, termed "va" for virulent strain associated. The va proteins of B31 passages 1, 27, and 47 differed strikingly, thus raising the possibility that these changes may relate in a causal way to the differences in induction of protective immunity observed.

Some physical and microstructural properties of genipin-crosslinked gelatin-maltodextrin hydrogels.

The physical properties and microstructure of gelatin-maltodextrin hydrogels fixed with genipin (GP) were investigated as a function of pH (3-7), maltodextrin (MD) (0-9%, w/w) and GP (0-10 mM levels), at a constant gelatin (G) concentration (10%, w/w). Network strength (elastic modulus, E) and swelling behavior were characterized by large deformation testing and by swelling index (SI). In general, network strength increased and swelling decreased at higher pH, MD and GP levels, except at pH 3, where E was independent of the GP concentration until approximately 7.5 mM, above which it declined. Confocal scanning laser microscopy (CLSM) images showed phase separation to be suppressed at pH 3, whereas at pH 7, separation into a self-similar dispersed phase was apparent. Overall, the judicious use of GP to crosslink G was an appropriate means of kinetically trapping MD within the gelatin network.

The gene for the S7 ribosomal protein of Chlamydia trachomatis: characterization within the chlamydial sfr operon.

The prokaryotic ribosomal operon, str, contains open reading frames for the two elongation factors, elongation factor G (EF-G) and elongation factor Tu (EF-Tu), and ribosomal proteins 57 and S12. The DNA sequence and predicted amino acid sequence for S7 from Chlamydia trachomatis are presented and compared with homologues from other prokaryotes. Also, the relationship of the S7 gene to the open reading frames for ribosomal protein S12 and EF-G is described. Significant amino acid homology is also noted when the amino-terminal sequence of chlamydial EF-G is compared with the cytoplasmic tetra-cycline resistance factors, tetM and tetO, from streptococci and Campylobacter jejuni. Related findings and possible resistance mechanisms for the newly recognized tetracycline-resistant clinical isolates of C. trachomatis are discussed.

Antibody to the retrovirus associated with the acquired immunodeficiency syndrome (AIDS). Presence in presumably healthy San Franciscans who died unexpectedly.

We performed autopsies and serologic tests in 189 subjects (152 men and 37 women) between 20 and 50 years of age with no history of immunosuppression who died unexpectedly and whose bodies were referred to the San Francisco coroner's office. Forty-eight of the 88 single men for whom addresses were available lived in areas of the city with a high incidence of the acquired immunodeficiency syndrome (AIDS). In addition, 36 of the subjects (30 men) were intravenous drug abusers. Antibody to the retrovirus associated with AIDS was present in 23 (18%) of the 121 subjects whose sera were tested. However, neither pathologic nor laboratory manifestations of AIDS were present in any of the 189 subjects who underwent autopsy. These results suggest that antibody to the retrovirus is common but subclinical manifestations of AIDS are uncommon in San Francisco, a city where the incidence of clinical AIDS is high.

Use of the transbronchial biopsy for diagnosis of opportunistic pulmonary infections in acquired immunodeficiency syndrome (AIDS).

The authors used a method that includes Giemsa-stained touch preparations of a lung biopsy and culture of the same biopsy for viruses and microorganisms. The yield of etiologic agent diagnoses by this cytologic method was compared with yield from a simultaneous biopsy processed by usual histologic methods and stained by hematoxylin and eosin, silver methenamine, and other stains as necessary. Fifty-nine transbronchial biopsies and eight open lung biopsies were processed on 38 male patients with the clinical and laboratory features of acquired immunodeficiency syndrome (AIDS) to determine the etiology of their pulmonary disease. Pneumocystis carinii pneumonia was diagnosed in 16 patients. When sufficient material was available, the Giemsa-stained touch preparation agreed with the histologic examination. The touch preparation specimens were cultured, and seven of the patients with Pneumocystis carinii also had cytomegalovirus (CMV). Six additional patients had pulmonary CMV infection without evidence of Pneumocystis carinii pneumonia. Mycobacterial and fungal infections also were identified in these six patients. Sixteen patients had no specific diagnosis. With adequate material, their method provides reliable results and diagnosis within two to three hours of Pneumocystis carinii. In addition, it conserves the amount of specimen required, since the same material can be used for culture.

Blockade of CD40-CD154 interferes with human T cell engraftment in scid mice.

Antibodies to the ligand for CD40 (CD154) have been shown to exert profound effects on the development of cell-mediated immune responses in mice. The present study shows that an antibody to human CD154 (hCD40L) inhibits in vivo Tetanus toxoid (TT) specific secondary antibody responses in hu-PBL-scid mice, as well as the expansion of xenoreactive human T cells in the scid mice. A possible cause for the reduced expansion of xenoreactive, human T cells, was the decreased expression of murine B7.1 and B7.2 caused by the administration of anti-hCD40L. Therefore, it may be that defective maturation of murine antigen-presenting cells impeded the priming and expansion of human xenoreactive T cells.

Analysis of genomic DNA from Chlamydia trachomatis for Dam and Dcm methylation.

Chlamydia trachomatis is a Gram-negative eubacterium with a dimorphic developmental cycle and obligate intracellular growth in the eucaryotic host. The Dam transmethylase of Escherichia coli methylates at the N6 position of adenine in the sequence 5'-GATC-3' and the Dcm transmethylase adds methyl groups to the C5 position of the internal cytosines in the sequences 5'-CCWGG-3'. In contrast to E. coli, C. trachomatis DNA appears to have unmethylated Dam sites and only low level Dcm methylation.

A comparison of the early inflammatory effects of an agr-/sar- versus a wild type strain of Staphylococcus aureus in a rat model of endophthalmitis.

PURPOSE: We examined the ability of a wild type and an isogenic mutant strain of Staphylococcus aureus, deficient in the production of hemolysins and lipase (agr (-)/sar (-)), to induce endophthalmitis and inflammatory cell infiltration into the eye at 6, 24 and 48 hours after injection in a rat model of endophthalmitis. METHODS: Rat eyes were injected with 25 microl of viable S. aureus or sterile saline. Eyes were graded for clinical signs of inflammation daily, removed and processed for standard histologic analysis 6, 24 and 48 hours after injections. Comparisons of clinical scores and mean inflammatory cell numbers were made between S. aureus and control injected eyes. RESULTS: Both experimental groups developed clinical signs of endophthalmitis and demonstrated infiltration of inflammatory cells at 24 and 48 hours. Clinical inflammation in the Mutant I group was less than the wild type group at these times and significantly less at 48 hours (p<0.05). No statistically significant difference in the number of inflammatory cells was detected between the wild type and Mutant I injected eyes at 24 hours. At 48 hours, inflammatory cells increased by 75.0% in the wild type group and decreased by 19.0% in the Mutant I group and a statistically significant difference was seen between these two groups (p<0.05). At all times, the majority of inflammatory cells were neutrophils. By 48 hours, an increase in monocytes-macrophages was noted. CONCLUSION: Both strains of S. aureus induced clinical signs of inflammation and inflammatory cell infiltration. Clinical inflammation and inflammatory cell numbers were less in rats injected with the Mutant I strain. These results suggest that hemolysins and lipase may be important in the early induction phase of the inflammatory response.

Microbial evaluation: 139 implants removed from symptomatic patients.

Possible adverse effects of microbial organisms have been implicated in symptomatic silicone implant patients. In the literature, numerous authors have investigated the possible role of infection with respect to implant problems. To date, various bacterial species have been reported, including Staphylococcus aureus, Staphylococcus epidermidis, peptostreptococci, and Clostridium perfringens. Infections in polyurethane-coated prostheses also have been shown to prolong morbidity. Antibiotic use has been relatively empirical in this regard. The purpose of this study was, first, to determine the frequency, type, and clinical relevance of microbial colonization on implant surfaces removed from symptomatic patients and, second, to determine possible effects of microbial colonization on implant integrity (gel bleed, rupture). A total of 139 implants from 72 symptomatic patients were entered into the prospective clinical study between February of 1993 and July of 1994 at the UCLA Medical Center. The implant shell types included smooth (79 percent), polyurethane (8 percent), textured (7 percent), and smooth and Dacron (6 percent). The implant locations were subglandular (71 percent), submuscular (28 percent), and subcutaneous (1 percent). Of the 139 implants removed, 69 percent were intact and 31 percent were ruptured. Forty-seven percent of 139 implants were culture-positive. Propionibacterium acnes was isolated most frequently (57.5 percent), followed by Staphylococcus epidermidis (41 percent), and then Escherichia coli (1.5 percent). No fungal infections were identified. Culture positivity was not significantly associated with systemic symptoms. Sixty-seven percent of the positive culture implants were intact; 33 percent were ruptured. The frequency (47 percent) and types (P. acnes and S. epidermidis) of microbial colonization are determined in symptomatic silicone implant patients.

Direct hybridization and amplification applications for the diagnosis of infectious diseases.

We are entering an exciting new era of molecular diagnostics in the clinical microbiology laboratory. A number of perspectives are presented in this review. First presented was a discussion of molecular diagnostics for detection of the bacterium, Chlamydia trachomatis. This is especially relevant since the tests available for this organism represent the forefront of commercial systems. Also, these tests exemplify the difficulties and advantages inherent to future molecular diagnostics for all types of disease processes. Next, a discussion of the techniques thus far employed in the field of clinical microbiology is presented. Obvious overlap exists with other areas of molecular pathology. However, the emphasis is on which techniques have proven most useful in identifying infectious agents. Finally, the features of a successful clinical microbiology diagnostics laboratory are presented, including test component requirements, laboratory personnel, quality assurance techniques, and physical laboratory setting. It is hoped that helpful advice and references are provided that will assist individual clinical laboratories as they enter the field of molecular diagnostics of infectious diseases.

Defining the unknown: molecular methods for finding new microbes.

The purpose of this article is to describe specific applications of molecular diagnostics that are currently identifying suspected or unidentified microbial pathogens. The techniques reviewed include (i) the use of specific primers and PCR to identify new microbes, (ii) PCR amplification of conserved 16S rRNA sequence with subsequent identification of specific internal sequence from the candidate bacterial pathogen, and (iii) an exciting new modification of PCR called RDA or "reverse PCR" that can identify unique infectious agents in diseased tissue. The field will continue to expand rapidly and, it is hoped, contribute to a better understanding of the microbial environment with which humans coexist. Also, molecular techniques will eventually be applied in the demonstration of pathogenesis by the various newly identified microbial pathogens.

Developmental-form-specific DNA-binding proteins in Chlamydia spp.

We identified DNA-binding proteins specific to the elementary body (EB) developmental form of Chlamydia spp. Chlamydial proteins from whole lysates were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with nick-translated chlamydial DNA. By this method, C. trachomatis serovar L2 EBs had three unique protein bands of 58,000, 25,700, and 17,000 molecular weight not seen in the reticulate bodies. The 17,000-molecular-weight protein and the 25,700-molecular-weight protein were identified in an acid-soluble protein fraction and were resistant to high-salt elution, similar to other procaryotic nucleoproteins. The 17,000-molecular-weight protein was also detected in preparations with isolated chromosomes from EBs. Preliminary characterization indicated that the protein-DNA interaction was principally charge related.

The gene for the S7 ribosomal protein of Chlamydia trachomatis: characterization within the chlamydial str operon.

The prokaryotic ribosomal operon, str, contains open reading frames for the two elongation factors, elongation factor G (EF-G) and elongation factor Tu (EF-Tu), and ribosomal proteins S7 and S12. The DNA sequence and predicted amino acid sequence for S7 from Chlamydia trachomatis are presented and compared with homologues from other prokaryotes. Also, the relationship of the S7 gene to the open reading frames for ribosomal protein S12 and EF-G is described. Significant amino acid homology is also noted when the amino-terminal sequence of chlamydial EF-G is compared with the cytoplasmic tetracycline resistance factors, tetM and tetO, from streptococci and Campylobacter jejuni. Related findings and possible resistance mechanisms for the newly recognized tetracycline-resistant clinical isolates of C. trachomatis are discussed.

Developmental regulation of tandem promoters for the major outer membrane protein gene of Chlamydia trachomatis.

Chlamydia trachomatis has a biphasic developmental cycle which is characterized by qualitative and quantitative changes in protein expression. The molecular mechanisms that mediate these changes are unknown. Evidence for transcriptional regulation of the chlamydial major outer membrane protein gene (omp1) was found by Northern hybridization of RNA isolated sequentially during the chlamydial developmental cycle. Early in the growth cycle a single transcript was detected, which was followed hours later in the cycle by an additional transcript. Mapping of the initiating nucleotide for each transcript suggested that this gene is regulated by differential transcription from tandem promoters.

A cumulative experience examining the effect of natural and synthetic antimicrobial peptides vs. Chlamydia trachomatis.

We tested the activity of 48 structurally diverse antimicrobial peptides against Chlamydia trachomatis, serovar L2. The peptides' activity against C. trachomatis, serovar L2 was measured in 48-h McCoy cell shell vial assays. Peptides of 16-20 amino acids were more active than larger peptides, such as defensins. Beta-sheet protegrins, as well as alpha-helical peptides such as novispirin (G-10) were equally active. Enantiomers were as active as native structures. Moderate-sized circular mini-defensins were less effective against C. trachomatis. Moderate-sized cationic peptides may be useful in microbicide preparations designed to prevent chlamydial infection.

Differential human serologic response to two 60,000 molecular weight Chlamydia trachomatis antigens.

Chlamydia trachomatis causes sexually transmitted diseases and is associated with serious long-term sequelae such as tubal infertility and ectopic pregnancy. There have been suggestions that chlamydial antigens of approximately 57,000-60,000 Mr may be involved in the immunopathology. Two important chlamydial antigens of 57,000-60,000 Mr are a Triton X-100-soluble antigen, which induces hypersensitivity in ocular models, and a sarcosyl-insoluble cysteine-rich structural protein, omp2. In this study, a 57,000 Mr Triton X-100-soluble protein was characterized as the chlamydial homolog of groEL, a heat shock protein. Using protein fractions, antibody responses of pelvic inflammatory disease (PID) and ectopic pregnancy patients to chlamydial groEL and omp2 were differentiated. Nearly all patients in both groups were reactive to omp2. Of those with titers greater than or equal to 1:512, 31% of PID sera and 81% of ectopic pregnancy sera were positive for chlamydial groEL (P = .004). This selectivity suggests that women with PID who develop chronic sequelae are those with antibody to groEL.

Diversity of Chlamydia trachomatis major outer membrane protein genes.

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (omp1) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of omp1 gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.

Protegrins: structural requirements for inactivating elementary bodies of Chlamydia trachomatis.

We tested 20 protegrins against Chlamydia trachomatis serovar L2 (L2/434/Bu). Five of the protegrins had native structures; the others included nonamidated, enantiomeric, and truncated variants and peptides with <2 disulfide bonds. Antichlamydial activity resided principally in residues 5 to 15 of native protegrin PG-1, and optimal activity required both intramolecular disulfide bonds.