In vivo study of the initial bacterial adhesion on different implant materials.

OBJECTIVE: Biofilm formation on implant materials plays a major role in the aetiology of periimplantitis. The aim of this study was to examine in vivo the initial bacterial adhesion on six different implant materials. METHODS: The implant materials Ti-m, TiUnite®, ZiUnite®, ATZ-m, ATZ-s, TZP-A-m were tested using bovine enamel slabs as controls. All materials, fixed on splint systems, were examined after 30 min and 120 min of oral exposure. DAPI staining was used for quantitative analysis of the initially adherent microorganisms. Initial adherent microorganisms were visualised by fluorescence In situ-hybridisation (FISH) and quantified by confocal laser scanning microscopy (CLSM). The targets of the oligonucleotide probes were Eubacteria, Veillonella spp., Fusobacterium nucleatum, Actinomyces naeslundii and Streptococcus spp. RESULTS: DAPI analysis showed that increasing the time of oral exposure resulted in an increasing amount of initial adherent bacteria. The highest level of colonisation was on ZiUnite®, with the lowest occurring on the bovine enamel, followed by Ti-m. This early colonisation correlated significantly with the surface roughnesses of the materials. FISH and CLSM showed no significant differences relating to total bacterial composition. However, Streptococcus spp. was shown to be the main colonisers on each of the investigated materials. CONCLUSION: it could be shown that within an oral exposure time of 30 min and 120 min, despite the salivary acquired pellicle initial biofilm formation is mainly influenced directly or indirect by the material surface topography. Highly polished surfaces should minimise the risk of biofilm formation, plaque accumulation and possibly periimplantitis.

Airborne microbes in different dental environments in comparison to a public area.

OBJECTIVE: The aim of this study was to investigate quantitatively and qualitatively the airborne microbial load in a multi-chair dental clinic, a normal dental practice and a non-dental public area over a time period of four days and at different time points to estimate the risk of infections during dental surgery. METHODS: A multi-chair and a single chair treatment room each were examined in comparison to a non-medical public area over a period of four days. The colony forming units m(-3) (CFUs) were determined and isolated bacteria were characterised by morphological and biochemical analysis, gas chromatography and by 16S rRNA-gene sequencing. In the analyses enterococci were selectively searched for. RESULTS: The CFUs in the multi-chair treatment room were between 20 and 1050 CFU m(-3). During treatment the maxima reached were below 800 CFU m(-3). The values in the dental practice were between 200 and 600 CFU m(-3) and remain slightly but not significantly below the levels of the clinic (p > 0.05). In the common area, the CFUs were between 200 and 800 CFU m(-3). The proportion of micrococci was 56.8% in the clinic, 56.07% in the practice and 69.67% in the public area Coagulase-negative staphylococci constituted 35% at the dental clinic, 25% at the bank and 38% at the dental practice. No significant differences amongst the units were detected in the microbial composition of their dental aerosols (p > 0.05). CONCLUSION: Although, the bacterial counts in dental room were not significantly higher than the bacterial counts in a public area, the risk from dental clinic might be higher than a public area due to the type of micro-organisms, host susceptibility and the exposure time.

Comparison of bacterial adhesion and cellular proliferation on newly developed three-dimensional scaffolds manufactured by rapid prototyping technology.

Scaffolds used in the field of tissue engineering should facilitate the adherence, spreading, and ingrowth of cells as well as prevent microbial adherence. For the first time, this study simultaneously deals with microbial and tissue cell adhesion to rapid prototyping-produced 3D-scaffolds. The cell growth of human osteosarcoma cells (CAL-72) over a time period of 3-11 days were examined on three scaffolds (PLGA, PLLA, PLLA-TCP) and compared to the adhesion of salivary microorganisms and representative germs of the oral flora (Porphyromonas gingivalis, Prevotella nigrescens, Candida albicans, Enterococcus faecalis, Streptococcus mutans, and Streptococcus sanguinis). Scanning electron microscopy (SEM), cell proliferation measurements, and determination of the colony forming units (CFU) were performed. The cell proliferation rates on PLLA and PLLA-TCP after 3, 7, and 11 days of cultivation were higher than on PLGA. On day 3 the proliferation rates on PLLA and PLLA-TCP, and on day 5 on PLLA-TCP, proved to be significantly higher compared to that of the control (culture plate). The strain which showed the most CFUs on all of the investigated scaffolds was P. gingivalis, followed by E. faecalis. No significant CFU differences were determined examining P. gingivalis among the biomaterials. In contrast, E. faecalis was significantly more adherent to PLGA and PLLA compared to PLLA-TCP. The lowest CFU values were seen with C. albicans and P. nigrescens. Salivary born aerobic and anaerobic microorganisms adhered significantly more to PLGA compared to PLLA-TCP. These results supported by SEM point out the high potential of PLLA-TCP in the field of tissue engineering.

Biofilm formation and composition on different implant materials in vivo.

Biofilm formation was evaluated on the following titanium and zirconia implants in vivo: machined titanium (Ti-m), modified titanium (TiUnite), modified zirconia (ZiUnite), machined alumina-toughened zirconia (ATZ-m), sandblasted alumina-toughened zirconia (ATZ-s), and machined zirconia (TZP-A-m). Bovine enamel slabs were used as controls. Surface morphologies were examined by atomic force (AFM) and scanning electron microscopy (SEM). The surface wettability was also determined. Twelve healthy volunteers wore a splint system with the tested materials. After 3 and 5 days the materials were examined by fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). The levels of Streptococcus spp., Veillonella spp., Fusobacteriaum nucleatum, and Actinomyces naeslundii were quantitatively determined. The biofilm thickness was found to be between 19.78 and 36.73 µm after 3 days and between 26.11 and 32.43 µm after 5 days. With the exception of Ti-m the biofilm thickness after 3 days was correlated with surface roughness. In addition to Streptococcus spp. as the main component of the biofilm (11.23-25.30%), F. nucleatum, A. naeslundii, and Veillonella spp. were also detected. No significant differences in biofilm composition on the implant surfaces could be observed. In total, the influence of roughness and material on biofilm formation was compensated by biofilm maturation.

Tissue engineering of composite grafts: Cocultivation of human oral keratinocytes and human osteoblast-like cells on laminin-coated polycarbonate membranes and equine collagen membranes under different culture conditions.

In complex craniomaxillofacial defects, the simultaneous reconstruction of hard and soft tissue is often necessary. Until now, oral keratinocytes and osteoblast-like cells have not been cocultivated on the same carrier. For the first time, the cocultivation of human oral keratinocytes and human osteoblast-like cells has been investigated in this study. Different carriers (laminin-coated polycarbonate and equine collagen membranes) and various culture conditions were examined. Human oral keratinocytes and human osteoblast-like cells from five patients were isolated from tissue samples, seeded on the opposite sides of the carriers and cultivated for 1 and 2 weeks under static conditions in an incubator and in a perfusion chamber. Proliferation and morphology of the cells were analyzed by EZ4U-tests, light microscopy, and scanning electron microscopy. Cocultivation of both cell-types seeded on one carrier was possible. Quantitative and qualitative growth was significantly better on collagen membranes when compared with laminin-coated polycarbonate membranes independent of the culture conditions. Using perfusion culture in comparison to static culture, the increase of cell proliferation after 2 weeks of cultivation when compared with the proliferation after 1 week was significantly lower, independent of the carriers used. In conclusion, the contemporaneous cultivation of human oral keratinocytes and human osteoblast-like cells on the same carrier is possible, a prerequisite for planned in vivo studies. As carrier collagen is superior to laminin-coated polycarbonate membranes. Regarding the development over time, the increase of proliferation rate is lower in perfusion culture. Examinations of cellular differentiation over time under various culture conditions will be subject of further investigations.

Bacterial and Candida albicans adhesion on rapid prototyping-produced 3D-scaffolds manufactured as bone replacement materials.

Rapid prototyping (RP)-produced scaffolds are gaining increasing importance in scaffold-guided tissue engineering. Microbial adhesion on the surface of replacement materials has a strong influence on healing and long-term outcome. Consequently, it is important to examine the adherence of microorganisms on RP-produced scaffolds. This research focussed on manufacturing of scaffolds by 3D-bioplotting and examination of their microbial adhesion characteristics. Tricalciumphosphate (TCP), calcium/sodium alginate, and poly(lactide-co-glycolic acid) (PLGA) constructs were produced and used to study the adhesion of dental pathogens. Six oral bacterial strains, one Candida strain and human saliva were used for the adhesion studies. The number of colony forming units (CFU) were determined and scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were performed. Microorganisms adhered to all scaffolds. All strains, except for Streptococcus oralis, adhered best to PLGA scaffolds. Streptococcus oralis adhered to each of the biomaterials equally. Streptococcus mutans and Enterococcus faecalis adhered best to PLGA scaffolds, followed by alginate and TCP. Prevotella nigrescens, Porphyromonas gingivalis, Streptococcus sanguis, and Candida albicans showed the highest adherence to PLGA, followed by TCP and alginate. In contrast, the microorganisms of saliva adhered significantly better to TCP, followed by PLGA and alginate. SEM observations correlated with the results of the CFU determinations. CLSM detected bacteria within deeper sheets of alginate. In conclusion, because of the high adherence rate of oral pathogens to the scaffolds, the application of these biomaterials for bone replacement in oral surgery could result in biomaterial-related infections. Strategies to decrease microbial adherence and to prevent infections due to oral pathogens are discussed.

Comparative in vitro study of the cell proliferation of ovine and human osteoblast-like cells on conventionally and rapid prototyping produced scaffolds tailored for application as potential bone replacement material.

Reconstruction of bone defects in the field of craniomaxillofacial surgery is a relevant problem. In regenerative medicine, autologous bone is not available sufficiently. The full replacement of autologous bone grafts is required. A promising research field is the bone engineering. Especially the application of rapid prototyping (RP) enables new perspectives concerning the scaffold design. The aim of the study was to compare scaffolds produced by RP-technology (native and plasma-coated PLGA-scaffolds) with conventionally produced scaffolds (agar plates with hydroxyapatite and hyaluronic acid coated agar plates with hydroxyapatite) relating to proliferation, adhesion, and morphology of osteoblasts to get knowledge about the application potential of such 3D-manufactured matrices for bone engineering. TissueFoil E served as reference. To compare the scaffolds, 12 ovine and 12 human osteoblast-like cell cultures of the skull were used. Results were obtained by EZ4U, scanning electron microscopy, and light microscopy. The highest cell proliferation rate of human osteoblast-like cells was measured on TissueFoil E followed by plasma-coated PLGA-scaffolds and uncoated PLGA-scaffolds, whereas of ovine osteoblast-like cells on plasma-coated PLGA-scaffolds followed by TissueFoil E and uncoated PLGA-scaffolds. Human and ovine osteoblast-like cells on coated and uncoated agar plates had significant lower proliferation rates compared with TissueFoil E and PLGA-scaffolds. These results showed the potential of RP in the field of bone engineering. Mechanical properties of such scaffolds and in vivo studies should be investigated to examine if the scaffolds hold up the pressure it will undergo long enough to allow regrowth of bone and to examine the revascularization.

Growth of human osteoblast-like cells on beta-tricalciumphosphate (TCP) membranes with different structures.

Bioresorb, a bioactive, bioresorbable bone replacement material, consisting of pure beta-tricalciumphos phate (TCP) ceramic, was evaluated in cell culture with human osteoblast-like cells. The aim of our study was to investigate the influence of TCP on the growth behavior of human osteoblast-like cells. Different granule sizes and plate sizes were used in this study: Granule sizes 500-1000 microm, 1000-2000 microm and 2000-5000 microm; plate sizes 1.7 mm, 2.0 mm and 2.2 mm. Under scanning electron microscopic (SEM) observations the cell colonization on the surface of the biomaterial and the tissue compatibility were studied. Thin sections were used to study the growth of human osteoblast-like cells inside the biomaterial. It could be clearly shown that all investigated biomaterials are tissue compatible and that the size and structure (granule or plate) of the biomaterial effects the colonization rate. Bioresorb plates enhance the colonization comparable to granule. The results obtained by SEM and thin sections were confirmed immunhistochemically by the nonradioactive assay EZ4U - EASY FOR YOU.In conclusion, all investigated sizes and structures of Bioresorb are tissue compatible but the cell growth is much better on plates than on granule small in size. The results suggest that the plates may be favourable useful as scaffold for regrowth of bone.

Comparative in vitro study of the proliferation and growth of human osteoblast-like cells on various biomaterials.

In vitro studies about the growth behavior of osteoblasts onto biomaterials is a basic knowledge and a screening method for the development and application of scaffolds in vivo. In this in vitro study human osteoblast-like (HOB) cells were cultured on seven different biomaterials used in dental and craniomaxillofacial surgery, respectively. The tested biomaterials were synthetic biodegradable (MacroPore, Ethisorb, PDS, Beriplast P) and nonbiodegradable polymers (Palacos) as well as calcium phosphate cement (BoneSource) and titanium. The cell proliferation and cell colonization were analyzed by scanning electron microscopy and EZ4U-test. Statistical analysis were performed. HOB-like cells cultivated on Ethisorb showed the highest proliferation rate. The proliferation rate was statistically significant compared with Palacos, MacroPore, and BoneSource. Whereas, Beriplast, PDS, and titanium yielded lower proliferation rates. However, there was no statistically significant difference compared with Palacos, MacroPore, and BoneSource. SEM analysis showed no significant difference in individual cell features and cell colonization. But an infiltration and a growth of HOB-like cells throughout the porous structure of Ethisorb, which is formed by crossing fibers, is a striking different feature (macrotopography). This feature can explain the highest proliferation rate of Ethisorb. The results showed that HOB-like cells appear to be sensitive to substrate composition and topography. Moreover, the basis for further studies with such biomaterial/osteoblast constructs in vivo are provided. Further focusing points are developing techniques to fabricate three-dimensional porous biomaterial/cell constructs, studying the tissue reaction and the bone regeneration of such constructs compared with the use of autologous bone.

Structure and in vitro cytocompatibility of the gastropod shell of Helix pomatia.

Distinguishing features of biological constructions are high stability and adaptation to their environment. Beside biocompatibility, nontoxicity and degradability these characteristics are demanded for new biomaterials in the field of tissue engineering. This study investigated the chemical composition, the organization and the in vitro osteoconductive potential of the terrestrial gastropod shell (Helix pomatia) on CAL72 and human osteoblast-like cells. Chemical composition of the biomaterial was examined by X-ray diffraction (XRD) and scanning electron microscopy (SEM) was performed to analyze the architecture of the snail shell and the morphology of the seeded cells. A double staining procedure (FDA/PI) and a proliferation test (EZ4U) assessed the viability of the cells. Microscopical images showed the multilayered architecture of the aragonite shell with hexagonal crystals on the inner side. The cells spread well on the biomaterial and the highest proliferation rate could be measured with CAL72 cells on the inner shell surface. The osteoconductive effects of this natural biomaterial could encourage further experiments in the field of tissue engineering.

Differences in the fatty acid composition of KB-cells and gingival keratinocytes is culture medium additive dependent.

The influence of culture medium additives foetal bovine serum (FBS), serum effective substitutes (SES) and human autologous serum on the fatty acid profile of KB-cells and human gingival keratinocytes was examined. The KB-cells were cultivated in RPMI medium added with FBS or SES and the gingival keratinocytes in D-MEM added with FBS or human autologous serum. Two days before the cells were prepared for gas chromatography (GC), the media were changed to serum- and antibiotic-free media. Whole fatty acids of the cells were analysed using GC and the fatty acid profiles were compared. KB-cells as well as gingival keratinocytes changed their fatty acid composition, according to the medium additive used. Significant differences were observed. In the case of KB-cells cultivated with SES the fatty acid changes suggest an increase of the membrane fluidity. Corresponding and significant differences were observed with gingival keratinocytes cultivated in medium added with human autologous serum: the membrane fluidity of the gingival keratinocytes was increased. It is supposed that an increased membrane fluidity caused by a different fatty acid spectrum of the host cell may relate to mechanisms of bacterial adhesion. Consequently, in vitro studies on invasion and adhesion of bacteria or virus are dependent on the medium used. Further analyses are necessary of the functional effects caused by differences in the content of specific FAs, especially with regard to the application of cultivated cells in the field of tissue engineering.

Bacterial colonization on different suture materials--a potential risk for intraoral dentoalveolar surgery.

In this in vivo and in vitro study on resorbable (Monocryl and nonresorbable (Deknalon) monofilament sutures used in intraoral dentoalveolar surgery the bacterial colonization was compared. For the in vivo study the sutures were applied in 11 patients during dental surgery. Eight days postoperative the sutures were removed and the adhered bacteria were isolated and identified by biochemistry, morphology, antibiotic susceptibility, and gas chromatography. The colonization was studied by scanning electron microscopy. Aerobic and anaerobic bacteria were isolated in nearly equal colony-forming units (cfu) on each suture. In comparison with Monocryl about 15% more aerobic and anaerobic strains were isolated on Deknalon. Regarding the pathogens only, about three times more anaerobic strains were isolated on both sutures in total. Additionally, more pathogens were found on Deknalon than on Monocryl (aerobic >40%, anaerobic >25%). The variety of bacteria correspond with purulent infections, not with normal oral flora. Intraindividual comparisons of cfu showed differences in dependence of the patient as described for subgingivale plaques. For the in vitro study the sutures were incubated with Streptococcus intermedius and Prevotella intermedia for 0.5 h. Scanning electron microscopy was performed to examine qualitatively the level of bacterial adherence. After 0.5 h the bacteria adhered very well. The colonization rate of Streptococcus intermedius on both sutures was similar. Coccoid bacteria within biofilms were seen. The growth of Prevotella intermedia was much better on Deknalon than on Monocryl. The risk of bacteremia at the time of suture removal is discussed.

Search for ideal biomaterials to cultivate human osteoblast-like cells for reconstructive surgery.

In this study we cultured human osteoblast-like cells on 16 different biomaterials to find an optimal biomaterial for subsequent use in reconstructive surgery. The tested biomaterials can be divided into five groups: collagen-based membranes of bovine, equine or calf origin, tricalcium phosphate based membranes (alpha and beta), hyaluronic acid based, anorganic bovine bone and anorganic silicone-based membranes. Cell proliferation and cell colonization (Environmental Scanning Electron Microscope, ESEM) analysis were performed. The results of the study demonstrated that four of the examined biomaterial/cell constructs showed a very good proliferation rate and cell density: No. 3 (Tissue Vlies), No. 7 (Sepra film), No. 16 (Biobrane) and No. 17 (Biomend). No favourable group of biomaterials was noticeable. Moreover, the results indicate that these four biomaterials as a part of bone constructs are the best tools for engineering new bone tissue. In contrast, biomaterials No. 19a (Bio-Oss) and 19b (Bio-Oss Collagen) showed the lowest proliferation rates. The result of No. 19b was improved by treatment in the perfusion chamber for 48 h as well as by additional use of vacuum. The present study is an important base for further analysis of biomaterials and consequently for the development of tissue engineering.

How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials.

The optimization of seeding and culturing of human osteoblast-like cells on three collagen-based biomaterials (bovine, equine and calf collagen membrane) was studied by cell proliferation and cell colonization (scanning electron microscopy) analysis. Osteoblasts of five patients were seeded onto the three biomaterials and two different parameters were varied: the time intervals between initial seeding and adding culture medium (2 h 6 h. 12 h, 24 h) and the seeding concentration (1 x 10(5), 1 x 10(6), 2 x 10(6)cells/ml) of cells onto biomaterials. The results of the study demonstrated that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration effects the cell proliferation and the cell colonization. The best proliferation rate was achieved by adding the culture medium 2 h after initial seeding and with a seeding density of 1 x 10(5) cells/ml. Moreover, all three biomaterials resulted in different proliferation rates. The best proliferation rate resulted with the bovine collagen membrane. In conclusion, the examined parameters are very important for the development of the tissue engineering techniques and in a larger perspective also for reconstructive surgery.

The titanium surface texture effects adherence and growth of human gingival keratinocytes and human maxillar osteoblast-like cells in vitro.

The adhesion, orientation and proliferation of human gingival epithelial cells and human maxillar osteoblast-like cells in primary and secondary culture were studied on glossy polished, sandblasted and plasma-sprayed titanium surfaces by scanning electron microscopy and in thin sections. The primary cultured explants of human gingival epithelial cells attached, spread and proliferated on all titanium surfaces with the greatest extension on the polished and the smallest extension on plasma-sprayed surfaces. In secondary suspension cultures of gingival keratinocytes, attachment spreading and growth was only observed on polished and plasma-sprayed surfaces, but not on sandblasted surfaces. Moreover, the attachment of these cells depended on the seeding concentration as well as on the coating with fetal calf serum. Cells on polished surfaces developed an extremely flat cell shape, but on sandblasted and plasma-sprayed surfaces a more cuboidal shape. In contrast human maxillar osteoblasts seeded as secondary suspension cultures attached very well to all three differently textured titanium surfaces and showed identical growth patterns independent of the titanium surface structure. These findings suggest that cell morphology, orientation, proliferation and adhesion of human gingival epithelial cells in primary or secondary culture are dependent on the texture of the titanium surface whereas no such differences were observed for maxillar osteoblast-like cells. In conclusion, the soft tissue integration and response is more influenced by the surface texture than the process of osseointegration.

Immunohistochemical study during healing of free palatal mucosa grafts on plastic-embedded samples.

This immunohistochemical study describes changes in the histology and in the distribution of the basement membrane components laminin and collagen IV as well as of the cytokeratins (CK)1/2/10/11, CK5/6, CK13, CK14, CK17, CK19 during the take of free split mucosa (epithelial and connective tissue) transplants in humans up to 36 months post-operative. Histology showed a flattening of the epithelial layer within the first 2 weeks after grafting, followed initially by an increase (25-30 layers, week 6) and later on by a decrease of cell layers in the epithelium (15-20 layers, week 20). From that time onwards, clear stratification and reteridges as signs of differentiation were present. Up to day 14 of graft take, the linear staining patterns for laminin and collagen IV were interrupted, which was not observed at any later stage. During this early interval CK5/6, CK1/2/10/11, CK14 and CK17 were expressed in all epithelial layers. The reactions for CK5/6 and CK1/2/10/11 were less intensive. At 6 weeks, CK1/2/10/11 stained the intermediate and superficial layers, being consistent with findings after longer graft take. CK5/6 reacted in the basal and intermediate cell layers, and CK13, CK14 and CK17 reacted in all layers. In the following period up to week 20, CK5/6 were found in the parabasal cells of the intermediate cell layers and the basal cells. CK14 staining was confined to these cell layers too, but also showed some reaction in the superficial layers. CK13 and CK17 were still bound to all layers. At 7 months post-operative, CK5/6 and CK14 were seen in their typical localisation in the basal cell layer and the parabasal cells of the intermediate layers, CK17 was seen mainly in the intermediate layer and CK13 was seen in focal areas of all layers. Anti-CK19 reacted only with single basal and parabasal cells up to week 20. These results suggest that during healing of mucosal autografts there is a sequence of changes in the expression of cell biological differentiation markers that may involve an epithelio-connective tissue interaction before the typical patterns for the donor side were observed again on the gingiva or mucosa of the hard palate.

In vitro susceptibilities of Chlamydia pneumoniae isolates from German patients and synergistic activity of antibiotic combinations.

The susceptibilities of six Chlamydia pneumoniae type strains and of six German patient isolates to erythromycin, azithromycin, roxithromycin, clarithromycin, doxycycline, ofloxacin, and rifampin were investigated. MICs and minimal chlamydicidal concentrations were all within the ranges reported previously. Combinations of azithromycin with either ofloxacin, doxycycline, or rifampin, as well as combinations of three antibiotics (rifampin, azithromycin, and ofloxacin or doxycycline), showed synergistic activity against C. pneumoniae.

Reactions of polyclonal and neutralizing anti-p54 monoclonal antibodies with an isolated, species-specific 54-kilodalton protein of Chlamydia pneumoniae.

A recently described 54-kDa protein has been detected in six type strains and three patient isolates of Chlamydia pneumoniae by immunoblotting with sera from patients positive for antibodies to C. pneumoniae by the microimmunofluorescence test. This protein was not found in either C. trachomatis E or C. psittaci Z 432 as an antigen, confirming its species specificity. The 54-kDa protein was isolated by continuous-elution electrophoresis and immunoglobulin G monoclonal antibodies (MAbs) against the isolated antigen were produced. MAb 8B11E6 reacted only with the 54-kDa band of C. pneumoniae and not with C. trachomatis E or C. psittaci in a Western immunoblot assay. This antibody was purified and tested for neutralizing activity together with three additional anti-p54-active MAbs (8B11E6, 8B11B4, and 10F1C1). In Buffalo green monkey cells, all of the MAbs significantly reduced the infectivity of C. pneumoniae elementary bodies, whereas no neutralizing activity could be observed with C. trachomatis E or C. psittaci Z 432. These results not only confirm the species specificity of the 54-kDa protein but also indicate that this protein might play an important role in the pathogenesis of C. pneumoniae infection. Furthermore, the results suggest a possible protective role of anti-p54 antibodies in an adaptive immune response.

Immune response to Chlamydia trachomatis heat-shock protein in infertile female patients and influence of Chlamydia pneumoniae antibodies.

A total of 446 sera from 245 patients with primary or secondary infertility, all of whom were examined laparoscopically, 117 patients with Chlamydia trachomatis-positive cervical swabs, and 84 control persons (50 obstetric patients and 34 female blood donors) were tested for antibodies to Chlamydia trachomatis and to Chlamydia pneumoniae with the microimmunofluorescence (MIF) test. MIF test antibody rates were highest in patients with complete tubal occlusion (73%) and in patients with proven Chlamydia trachomatis infection (74%), whereas only 9 to 10% of the control group showed Chlamydia trachomatis antibodies. Reaction to the 60 kDa antigen of Chlamydia trachomatis, a heat-shock protein (hsp) analogue, has been suggested as a possible marker for the development of chronic sequelae after Chlamydia trachomatis infection. Immunoblot analysis of 222 sera (169 infertility patients, 20 antigen-positive patients, and 33 mothers) showed a significantly higher anti-hsp antibody rate in patients with complete tubal occlusion than in infertility patients with normal fallopian tubes (76% vs. 19%, p < 0.001). The presence of antibodies not only to Chlamydia trachomatis but also to Chlamydia pneumoniae in the MIF test was associated with a significantly higher rate of anti-hsp antibodies and with complete tubal occlusion. This association did not appear to be due to cross-reactivity between Chlamydia pneumoniae and Chlamydia trachomatis antibodies in the MIF test.