Intraoperative margin assessment during radical prostatectomy: is microscopy frozen in time or ready for digital defrost?

Intraoperative frozen section (IFS) is used with the intention to improve functional and oncological outcomes for patients undergoing radical prostatectomy (RP). High resource requirements of IFS techniques such as NeuroSAFE may preclude widespread adoption, even if there are benefits to patients. Recent advances in fresh-tissue microscopic digital imaging technologies may offer an attractive alternative, and there is a growing body of evidence regarding these technologies. In this narrative review, we discuss some of the familiar limitations of IFS and compare these to the attractive counterpoints of modern digital imaging technologies such as the speed and ease of image generation, the locality of equipment within (or near) the operating room, the ability to maintain tissue integrity, and digital transfer of images. Confocal laser microscopy (CLM) is the modality most frequently reported in the literature for margin assessment during RP. We discuss several imitations and obstacles to widespread dissemination of digital imaging technologies. Among these, we consider how the 'en-face' margin perspective will challenge urologists and pathologists to understand afresh the meaning of positive margin significance. As a part of this, discussions on how to describe, categorize, react to, and evaluate these technologies are needed to improve patient outcomes. Limitations of this review include its narrative structure and that the evidence base in this field is relatively immature but developing at pace.

Technologies and techniques to improve precision in breast conserving surgery.

Imprecision in breast conserving surgery results in high rates of take back to theatre for reexcision of margins. This paper reviews the various approaches to improving the precision of oncological margin control in breast conserving surgery. The review describes the rationale for improved tissue characterization over tumor localization and explores technology-free approaches, as well as progress being made to develop and test innovative technological solutions.

Lactoferrin Affects the Viability of Bacteria in a Biofilm and the Formation of a New Biofilm Cycle of Mannheimia haemolytica A2.

Respiratory diseases in ruminants are responsible for enormous economic losses for the dairy and meat industry. The main causative bacterial agent of pneumonia in ovine is Mannheimia haemolytica A2. Due to the impact of this disease, the effect of the antimicrobial protein, bovine lactoferrin (bLf), against virulence factors of this bacterium has been studied. However, its effect on biofilm formation has not been reported. In this work, we evaluated the effect on different stages of the biofilm. Our results reveal a decrease in biofilm formation when bacteria were pre-incubated with bLf. However, when bLf was added at the start of biofilm formation and on mature biofilm, an increase was observed, which was visualized by greater bacterial aggregation and secretion of biofilm matrix components. Additionally, through SDS-PAGE, a remarkable band of ~80 kDa was observed when bLf was added to biofilms. Therefore, the presence of bLf on the biofilm was determined through the Western blot and Microscopy techniques. Finally, by using Live/Dead staining, we observed that most of the bacteria in a biofilm with bLf were not viable. In addition, bLf affects the formation of a new biofilm cycle. In conclusion, bLf binds to the biofilm of M. haemolytica A2 and affects the viability of bacteria and the formation a new biofilm cycle.

Diagnosis and therapy of actinic keratosis.

Actinic keratosis (AK) is considered a chronic and recurring in situ skin neoplasia, with a possible transformation into invasive squamous cell carcinoma (SCC). Among others, predominant risk factors for development of AK are UV-light exposure and immunosuppression. Basal epidermal keratinocyte atypia (AK I) and proliferation (PRO score) seem to drive malignant transformation, rather than clinical appearance of AK (Olsen I-III). Due to the invasiveness of punch biopsy, those histological criteria are not regularly assessed. Non-invasive imaging techniques, such as optical coherence tomography (OCT), reflectance confocal microscopy (RCM) and line-field confocal OCT (LC-OCT) are helpful to distinguish complex cases of AK, Bowen's disease, and SCC. Moreover, LC-OCT can visualize the epidermis and the papillary dermis at cellular resolution, allowing real-time PRO score assessment. The decision-making for implementation of therapy is still based on clinical risk factors, ranging from lesion- to field-targeted and ablative to non-ablative regimens, but in approximately 85% of the cases a recurrence of AK can be observed after a 1-year follow-up. The possible beneficial use of imaging techniques for a non-invasive follow-up of AK to detect recurrence or invasive progression early on should be subject to critical evaluation in further studies.

Diagnose und Therapie aktinischer Keratosen: Diagnosis and therapy of actinic keratosis.

Actinic keratosis (AK) is considered a chronic and recurring in situ skin neoplasia, with a possible transformation into invasive squamous cell carcinoma (SCC). Among others, predominant risk factors for development of AK are UV-light exposure and immunosuppression. Basal epidermal keratinocyte atypia (AK I) and proliferation (PRO Score) seem to drive malignant turnover, rather than clinical appearance of AK (Olsen I-III). Due to the invasiveness of punch biopsy, those histological criteria are not regularly assessed. Non-invasive imaging techniques, such as optical coherence tomography (OCT), reflectance confocal microscopy (RCM) and line-field confocal OCT (LC-OCT) are helpful to distinguish complex cases of AK, Bowen's disease and SCC. Moreover, LC-OCT can visualize the epidermis and the papillary dermis at cellular resolution, allowing real-time PRO Score assessment. The decision-making for implementation of therapy is still based on clinical risk factors, ranging from lesion- to field-targeted and ablative to non-ablative regimes, but in approximately 85% of the cases a recurrence of AK can be observed after a 1-year follow-up. The possible beneficial use of imaging techniques for a non-invasive follow-up of AK to detect recurrence or invasive progression early on should be subject to critical evaluation in further studies.

Latanoprost-Loaded Nanotransfersomes Designed for Scalp Administration Enhance Keratinocytes Proliferation.

Latanoprost (LAT) has been shown to have a hypertrichotic effect, which makes it a promising candidate for alopecia treatments. For the first time, LAT has been encapsulated in nanotransfersomes in order to increase its efficacy. Ex vivo skin biodistribution was studied by confocal laser microscopy both in human scalp and pig skin. Results showed that nanotransfersomes increase the penetration of two different fluorochromes, with similar patterns in both species, compared with fluorochrome solutions containing no nanotransfersomes. Nanotransfersomes were stable under accelerated conditions (40 °C/75% RH) and long-term conditions (25 °C/60% RH) for up to 1 year, with no differences in vesicle size and polydispersity when LAT was loaded. Nanotransfersomes increased the LAT cell proliferation effect in HaCaT cell via MAPK signaling pathway. Collectively, our results demonstrate LAT-nanotransfersomes formulation could be a promising therapy for hair growth disorders.

Confocal laser microscopy for assessment of surgical margins during radical prostatectomy.

OBJECTIVE: To evaluate the feasibility of confocal laser microscopy (CLM) for intraoperative margin assessment as faster alternative to neurovascular structure-adjacent frozen-section examination (NeuroSAFE) during robot-assisted radical prostatectomy (RARP). PATIENTS AND METHODS: Surgical margins were assessed during 50 RARP procedures in patients scheduled for NeuroSAFE. Posterolateral sections were cut and imaged with CLM and further processed to conform with the NeuroSAFE protocol. Secondary resection (SR) was performed in case a positive surgical margin (PSM) was observed with NeuroSAFE. Afterwards, the CLM images were non-blinded assessed for the presence of PSMs. The accuracy of both NeuroSAFE and CLM was compared with conventional histopathology. Agreement for detection of PSMs between NeuroSAFE and CLM was evaluated with Cohen's kappa coefficient. Procedure times were compared with a Wilcoxon signed-ranks test. RESULTS: In total, 96 posterolateral sections of RP specimens were evaluated for the presence of PSMs. CLM identified 15 (16%) PSMs and NeuroSAFE identified 14 (15%) PSMs. CLM had a calculated sensitivity, specificity, positive predictive value and negative predictive value of 86%, 96%, 80% and 98% respectively for the detection of PSMs compared to definite pathology. After SR, residual tumour was found in six of 13 cases (46%), which were all identified by both techniques. There was a substantial level of agreement between CLM and NeuroSAFE (κ = 0.80). The median procedure time for CLM was significantly shorter compared to NeuroSAFE (8 vs 50 min, P < 0.001). The main limitation of this study was the non-blinded assessment of the CLM images. CONCLUSIONS: Compared to NeuroSAFE, CLM is a promising technique for intraoperative margin assessment and is able to reduce the time of intraoperative margin assessment.

Characterization of functional amyloid curli in biofilm formation of an environmental isolate Enterobacter cloacae SBP-8.

The biofilm formation by bacteria is a complex process that is strongly mediated by various genetic and environmental factors. Biofilms contribute to disease infestation, especially in chronic infections. It is, therefore important to understand the factors affecting biofilm formation. This study reports the role of a functional amyloid curli in biofilm formation at various abiotic surfaces, including medical devices, by an environmental isolate of Enterobacter cloacae (SBP-8) which has been known for its pathogenic potential. A knockout mutant of csgA, the gene encoding the major structural unit of curli, was created to study the effect of curli on biofilm formation by E. cloacae SBP-8. Our findings confirm the production of curli at 25 °C and 37 °C in the wild-type strain. We further investigated the role of curli in the attachment of E. cloacae SBP-8 to glass, enteral feeding tube, and foley latex catheter. Contrary to the previous studies reporting the curli production below 30 °C in the majority of biofilm-forming bacterial species, we observed its production in E. cloacae SBP-8 at 37 °C. The formation of more intense biofilm in wild-type strain on various surfaces compared to curli-deficient strain (ΔcsgA) at both 25 °C and 37 °C suggested a prominent role of curli in biofilm formation. Further, electron and confocal microscopy studies demonstrated the formation of diffused monolayers of microbial cells on the abiotic surfaces by ΔcsgA strain as compared to the thick biofilm by respective wild-type strain, indicating the involvement of curli in biofilm formation by E. cloacae SBP-8. Overall, our findings provide insight into biofilm formation mediated by curli in E. cloacae SBP-8. Further, we show that it can be expressed at a physiological temperature on all surfaces, thereby indicating the potential role of curli in pathogenesis.

The Antimicrobial Activity of Curcumin and Xanthohumol on Bacterial Biofilms Developed over Dental Implant Surfaces.

In search for natural products with antimicrobial properties for use in the prevention and treatment of peri-implantitis, the purpose of this investigation was to evaluate the antimicrobial activity of curcumin and xanthohumol, using an in vitro multi-species dynamic biofilm model including Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The antimicrobial activities of curcumin (5 mM) and xanthohumol (100 µM) extracts, and the respective controls, were evaluated with 72-h biofilms formed over dental implants by their submersion for 60 seconds. The evaluation was assessed by quantitative polymerase chain reaction (qPCR), confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). For the data analysis, comparisons were tested applying ANOVA tests with post-hoc Bonferroni corrections to evaluate the antimicrobial activity of both extracts. With qPCR, statistically significant reductions in bacterial counts were observed for curcumin and xanthohumol, when compared to the negative control. The results with CLSM and SEM were consistent with those reported with qPCR. It was concluded that both curcumin and xanthohumol have demonstrated antimicrobial activity against the six bacterial species included in the dynamic in vitro biofilm model used.

Antagonistic activity of endophytic actinobacteria from native potatoes (Solanum tuberosum subsp. tuberosum L.) against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum.

BACKGROUND: The native potatoes (Solanum tuberosum subsp. tuberosum L.) grown in Chile (Chiloé) represent a new, unexplored source of endophytes to find potential biological control agents for the prevention of bacterial diseases, like blackleg and soft rot, in potato crops. RESULT: The objective of this study was the selection of endophytic actinobacteria from native potatoes for antagonistic activity against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum, and their potential to suppress tissue maceration symptoms in potato tubers. This potential was determined through the quorum quenching activity using a Chromobacterium violaceaum ATCC 12472 Wild type (WT) bioassay and its colonization behavior of the potato plant root system (S. tuberosum) by means of the Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) targeting technique. The results showed that although Streptomyces sp. TP199 and Streptomyces sp. A2R31 were able to inhibit the growth of the pathogens, only the Streptomyces sp. TP199 isolate inhibited Pectobacterium sp. growth and diminished tissue maceration in tubers (p ≤ 0.05). Streptomyces sp. TP199 had metal-dependent acyl homoserine lactones (AHL) quorum quenching activity in vitro and was able to colonize the root endosphere 10 days after inoculation. CONCLUSIONS: We concluded that native potatoes from southern Chile possess endophyte actinobacteria that are potential agents for the disease management of soft rot and blackleg.

An easy and rapid staining method for confocal microscopic observation and reconstruction of three-dimensional images of echinoderm larvae and juveniles.

The morphologies of the internal organs of echinoderm larvae and juveniles are difficult to study using conventional optical microscopes because of their structural complexity and opaqueness. This paper describes an easy and rapid protocol involving Nile blue staining followed by benzyl alcohol/benzyl benzoate (BABB) clearing to overcome this limitation. This method was developed for a three-dimensional (3D) analysis of the internal structures of advanced larvae and juveniles of echinoderms (the sea lily Metacrinus rotundus, the sea urchin Hemicentrotus pulcherrimus, and the sand dollar Scaphechinus mirabilis) and is suitable for obtaining serial optical images by confocal microscopy without the use of specific antibodies or special reagents for labeling. Nile blue is an easy-to-use stain that offers several advantages for confocal microscopy such as it can stain various tissues with strong fluorescent signals without substantial bleaching during observation. We found that the strong fluorescence signal of Nile blue quickly yielded clear high-resolution optical section images for 3D reconstruction. BABB clearing rendered opaque larvae highly transparent. The clearing procedure was also easy and quick. During the process, agarose embedding prior to staining and clearing was found to be critical for handling the samples of less than 500-µm length and stabilizing their orientations. To conclude, the protocol described is useful for performing a rapid and accurate 3D morphological analysis of echinoderm larvae and juveniles.

[Confocal line-field OCT]. / Konfokale Line-Field-OCT.

Optical coherence tomography (OCT) and confocal laser microscopy (CLSM) are established non-invasive methods in clinical dermatological routine diagnosis. Whereas CLSM is especially useful to distinguish between nevi and melanoma, OCT is suitable for the diagnosis and differentiation of non-melanoma skin cancer. Line-field confocal optical coherence tomography (LC-OCT) is a new innovative device, which has better cellular resolution than OCT and a higher penetration depth than CLSM. Similar to CLSM, LC-OCT also allows 3D images in real time to be taken. Therefore LC-OCT is very useful for the examination of skin lesions of all kinds, since it unites the features of CLSM and OCT.

[Noninvasive diagnostic imaging in pediatric skin lesions]. / Nichtinvasive bildgebende Diagnostik bei Hauterkrankungen im Kindesalter.

Optical coherence tomography (OCT) and reflectance confocal laser microscopy (RCM) allow noninvasive imaging diagnostics of the skin. Since the indication for a biopsy in children is generally made cautiously, OCT and KLM can be helpful in the clarification of pediatric skin lesions. In addition, biopsies only represent a snapshot of a small area of the skin, while noninvasive methods allow several locations to be examined over time, so that dynamic changes and the course of therapy can also be determined. In pediatric dermatology, these diagnostic methods are particularly suitable for the clarification of atypical pigment lesions, for infectious dermatoses such as scabies or tinea, and for the diagnosis of inflammatory and degenerative dermatoses.

Evaluation of tissue-clearing techniques for intraorgan imaging of distribution of polymeric nanoparticles as drug carriers.

OBJECTIVE: The development of drug delivery systems using nanocarriers requires intraorgan imaging techniques for evaluating the distribution of nanocarriers. In this study, we evaluated the tissue-clearing techniques for the imaging of polymeric nanoparticles, a nanocarrier, in the liver used as a model of pigment-rich organ in mice. SIGNIFICANCE: The intraorgan imaging method of polymeric nanoparticles was examined without sectioning of organ samples for evaluating the delivery efficiency in preclinical studies. METHODS: DiI-loaded polymeric nanoparticles and fluorescence-tagged tomato lectin for fluorescence labeling of liver general structures were intravenously administered to mice. Tissue-clearing treatment of the mouse liver was performed using ClearT2, ScaleSQ(0), clearing agent comprising fructose, urea, and glycerol for imaging (FUnGI), clear unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and modified CUBIC techniques. Intraorgan fluorescence imaging in the liver was performed by confocal laser microscopy. RESULTS: ClearT2 treatment exhibited insufficient clearing capability in the mouse liver. Although CUBIC treatment exhibited the best clearing capability, the CUBIC caused DiI leakage. ScaleSQ(0), FUnGI, and modified CUBIC treatments exhibited better clearing capability than ClearT2 technique while preserving the DiI. In the fluorescence imaging, the CUBIC and modified CUBIC exhibited deeper visualization than with the ScaleSQ(0) and FUnGI; however, the CUBIC led to a change in DiI distribution. The modified CUBIC enabled the deepest visualization while preserving the distribution of DiI. CONCLUSION: The intraorgan imaging method was established using modified CUBIC technique by the intravenous administration of fluorescence-tagged tomato lectin for evaluating the distribution of polymeric nanoparticles in mouse pigment-rich organs.

Confocal laser microscopy as novel approach for real-time and in-vivo tissue examination during minimal-invasive surgery in colon cancer.

BACKGROUND: Histological analysis of surgical specimen is the gold standard for cancer classification. In particular, frozen histological diagnosis of vague peritoneal spots or uncertain excision of tumors plays a crucial role in the decision to proceed with or abandon an operation. Confocal laser microscopy (CLM) enables in-vivo and real-time high-resolution tissue analysis. This method has already been used during endoscopic assessments analyzing transformation of esophageal or colon mucosa. We examined whether a CLM device enables to distinguish between non-malignant and malignant tissue in vivo and real time and enables to assign peritoneal carcinomatosis spots to their primary tumor. In addition, we investigated whether the newly developed CLM camera device causes any tissue damage. METHODS: CC531 colon carcinoma cells were implanted on the serosa side of the colon and intraperitoneally in Wag/Rija rats via laparotomy. After 7 days of tumor growth, confocal laser microscopy in vivo was performed by re-laparotomy. Images of non-malignant and malignant tissue were characterized in terms of specific signal pattern. No fluorescent dye was used. Correlations to findings in conventional histology were systematically recorded and described. Potential tissue damage was examined by conventional histology. RESULTS: All animals survived the operative procedure and could be evaluated 7 days following surgery. No unexpected death occurred after surgery. Non-malignant colon is defined by small cycles of the microvilli of the colon. There is repetitive deregulated structure in colon carcinoma. Peritoneal carcinomatosis showed the same structural pattern as in primary colon carcinoma. In all examined cases, it was possible to differentiate between peritoneal carcinomatosis spots and non-malignant peritoneum. The CLM device did not cause any tissue damage. CONCLUSIONS: The CLM camera device reported here is feasible to identify peritoneal carcinomatosis spots, assign these spots to the primary tumor, as well as distinguish between non-malignant and malignant tissue in without using any fluorescent dye.

Bacterial biofilm formation is variably inhibited by different formulations of antibiotic-loaded bone cement in vitro.

PURPOSE: The aim of the present study was to quantitatively assess biofilm growth on the surface of bone cements discs containing different antibiotics, including colistin and linezolid. Biofilms of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Staphylococcus epidermidis were grown on bone cement discs for 96 h. METHODS: Biofilm amounts were measured by confocal laser microscopy using live/dead staining and dedicated software at different time intervals (48, 72, and 96 h). RESULTS: Bone cement containing vancomycin was not effective at reducing MRSA biofilm formation 96 h following bacterial inoculation. At a comparable time interval, linezolid-, clindamycin-, and aminoglycoside-loaded cement was still active against this biofilm. At the 72- and 96-h observations, S. epidermidis biofilm was present only on tobramycin and gentamicin discs. P. aeruginosa biofilms were present on cement discs loaded with colistin at all time intervals starting from the 48-h observation, whereas no biofilms were detected on tobramycin or gentamicin discs. CONCLUSION: Bone cements containing different antibiotics have variable and time-dependent windows of activity in inhibiting or reducing surface biofilm formation. The effectiveness of bone cement containing vancomycin against MRSA biofilm is questionable. The present study is clinically relevant, because it suggests that adding the right antibiotic to bone cement could be a promising approach to treat periprosthetic infections. Indeed, the antibiofilm activity of different antibiotic-loaded bone cements could be preoperatively assessed using the current methodology in two-stage exchange procedures.

Confocal and dermoscopic features of basal cell carcinoma in Gorlin-Goltz syndrome: A case report.

Gorlin-Goltz (GS) syndrome is an autosomal dominant disease linked to a mutation in the PTCH gene. Major criteria include the onset of multiple basal cell carcinoma (BCC), keratocystic odontogenic tumours in the jaws and bifid ribs. Dermoscopy and reflectance confocal microscopy represent imaging tools that are able to increase the diagnostic accuracy of skin cancer in a totally noninvasive manner, without performing punch biopsies. Here we present a case of a young woman in whom the combined approach of dermoscopy and RCM led to the identification of multiple small inconspicuous lesions as BCC and thus to the diagnosis of GS syndrome.

Determining Optimal Fluorescent Agent Concentrations in Dental Adhesive Resins for Imaging the Tooth/Restoration Interface.

Fluorescent dyes like Rhodamine B (RB) have been used to identify the spatial distribution of adhesive restorative materials in the tooth/restoration interface. Potential effects of the addition of RB to dental adhesives were addressed in the past, but no further information is available on how to determine suitable concentrations of RB in these bonding agents for imaging in the confocal laser scanning microscope. This study provides systematical strategies for adding RB to viscous dental adhesive resins, focusing on the determination of the lowest range of dye concentrations necessary to achieve an acceptable image of the dentin/adhesive interface. It was demonstrated that optimized images of the resin distribution in dentin can be produced with 0.1-0.02 mg/mL of RB in the (tested) adhesives. Our approaches took into account aspects related to the dye concentration, photophysical parameters in different host media, specimen composition and morphology to develop a rational use of the fluorescent agent with the resin-based materials. Information gained from this work can help optimize labeling methods using dispersions of low-molecular-weight dyes in different monomer blend systems.

[Nuclear delivery of oligonucleotides via nanocomposites based on TiO2 nanoparticles and polylysine].

The nuclear delivery of nucleic acid derivatives is an essential prerequisite for successful antisense therapy. Using laser confocal and electron microscopy, we have studied the uptake of fluorescently labeled oligonucleotides in the form of nanocomposites with polylysine and TiO2 nanoparticles into Caco2, MDCK, and HeLa cells. In all three cell lines, bright fluorescence has been detected after 30 min in the nuclei (excluding the nucleoli) of the interphase cells; no substantial increase in the intensity of the signal was observed for next 24 hours. In all cells undergoing mitosis, the signal was localized in the cytoplasm with zones of higher intensity around chromatin. In some cells, at the beginning of interphase (G-1 phase), fluorescence was not detected at all. The latter may be explained by the brief moment in the cell cycle when oligonucleotides delivered in the nanocomposite cannot be taken up by cells. The studied nanocomposites are prone to aggregation. The degree of aggregation increases with the storage time up to complete loss of the ability of the nanocomposites to penetrate the cells.

Paracoccidioides brasiliensis interacts with dermal dendritic cells and keratinocytes in human skin and oral mucosa lesions.

Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. In PCM the skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to immunohistochemical and/or immunofluorescence staining with anti-factor XIIIa (marker of dermal dendrocytes), anti-CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti-P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed for better visualization of the interaction between keratinocytes and the fungi. In sum, 42% of oral mucosa samples displayed yeast forms in Factor XIIIa dermal dendrocytes cytoplasm. Langerhans cells in skin and oral mucosa samples did not show yeast cells in their cytoplasm. In sum, 54% of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms. Factor XIIIa dendrocytes and keratinocytes may be acting as antigen-presenting cells to fulfill the probably impaired function of Langerhans cells in skin and oral mucosa of human PCM.