Evaluation of the biocompatibility of fish skin collagen with the mesenchymal stem cells in in vitro cultures.

There are scarce published data suggesting, that collagen extracted from fish skin may be an attractive alternative to mammalian-derived collagen for the in vitro cell cultures. In this study, we investigated proliferation potential and differentiation capability into osteogenic and adipogenic lineages of rat adipose-derived mesenchymal stem cells (rASCs) and human adipose-derived mesenchymal stem cells (hASCs) cultured on collagen extracted from silver carp and African sharptooth catfish skins, compared with commercially available mammalian collagen and collagen-free culture dishes. Our results revealed no significant differences between fish collagen and mammalian collagen in supporting cell viability and proliferation capacity. Fish-derived collagen is a cheap material derived from production waste, does not contain transmissible pathogens of mammalian origin, supports human cell cultures at comparable level to conventional collagen sources, and may be considered as the product of choice for the in vitro cell cultures.

Effect of adipose-derived stem cells seeding and surgical prefabrication on composite scaffold vascularization.

The study aimed to evaluate an angiogenic effect of adipose-derived stem cells (ASCs) seeding and surgical prefabrication (placing a vascular pedicle inside the scaffold) on developed composite scaffolds made of poly-ε-caprolactone (PCL), ß-tricalcium phosphate (ß-TCP), and poly (lactic-co-glycolic acid) (PLGA) (PCL+ß-TCP+PLGA). Moreover, we aimed to compare our data with previously tested PCL scaffolds to assess whether the new material has better angiogenic properties. The study included 18 inbred male WAG rats. There were three scaffold groups (six animals each): with non-seeded PCL+ß-TCP+PLGA scaffolds, with PCL+ß-TCP+PLGA scaffolds seeded with ASCs and with PCL+ß-TCP+PLGA scaffolds seeded with ASCs and osteogenic-induced. Each rat was implanted with two scaffolds in the inguinal region (one prefabricated and one non-prefabricated). After 2 months from implantation, the scaffolds were explanted, and vessel density was determined by histopathological examination. Prefabricated ASC-seeded PCL+ß-TCP+PLGA scaffolds promoted greater vessel formation than non-seeded scaffolds (19.73 ± 5.46 vs 12.54 ± 0.81; p = .006) and those seeded with osteogenic-induced ASCs (19.73 ± 5.46 vs 11.87±2.21; p = .004). The developed composite scaffold promotes vessel formation more effectively than the previously described PCL scaffold.

The impact of electroconductive multifunctional composite nanofibrous scaffold on adipose-derived mesenchymal stem cells.

BACKGROUND: The development of tissue-engineered scaffolds with electrical properties is the primary motivation of novel regenerative medicine. Electroconductive scaffolds are designed to mimic the injured tissue environment's electrical properties and regulate cellular behavior - growth, proliferation, and differentiation - that could stimulate the injured nerve's regeneration. METHODS: We fabricated dedicated electroconductive scaffolds and customized an appropriate device with an external current supply to expose cells on the scaffold to electrical stimulation (ES). Next, we isolated rat adipose-derived stem cells (ASCs) and performed in vitro experiments that combine cells, an electroconductive scaffold, NGF (nerve growth factor), and ES (90 mV/mm, constant, for four days). Finally, we checked cellular activity as proliferation, viability, morphology, the neurogenic differentiation potential of ASCs, cell alignment, and karyotype. RESULTS: We observed that the electrical stimulation did not change the viability and chromosome stability of rat ASCs, but altered slightly proliferation compared to non-stimulated cells. The combined effect of a scaffold, NGF, and ES caused morphology changes and enhancement of ASCs neuronal differentiation as indicated in ßIII-tubulin expression, actin organization, and upregulation of neurogenic gene expression. CONCLUSIONS: We developed an electroconductive scaffold and customized device for in vitro study with many experimental variants. Based on our results, we presumed that the established study scheme - including an electroconductive scaffold, NGF and ES - is biocompatible and could guide ASCs to differentiate in neurogenic lineage, thus may be potentially applied in nerve injury regeneration.

Modified Histopathological Protocol for Poly-ɛ-Caprolactone Scaffolds Preserving Their Trabecular, Honeycomb-like Structure.

Poly-ɛ-caprolactone (PCL) is now widely studied in relation to the engineering of bone, cartilage, tendons, and other tissues. Standard histological protocols can destroy the carefully created trabecular and honeycomb-like architecture of PCL scaffolds, and could lead to scaffold fibers swelling, resulting in the displacement or compression of tissues inside the scaffold. The aim of this study was to modify a standard histopathological protocol for PCL scaffold preparation and evaluate it on porous cylindrical PCL scaffolds in a rat model. In 16 inbred Wag rats, 2 PCL scaffolds were implanted subcutaneously to both inguinal areas. Two months after implantation, harvested scaffolds were first subjected to µCT imaging, and then to histopathological analysis with standard (left inguinal area) and modified histopathological protocols (right inguinal area). To standardize the results, soft tissue percentages (STPs) were calculated on scaffold cross-sections obtained from both histopathological protocols and compared with corresponding µCT cross-sections. The modified protocol enabled the assessment of almost 10× more soft tissues on the scaffold cross-section than the standard procedure. Moreover, STP was only 1.5% lower than in the corresponding µCT cross-sections assessed before the histopathological procedure. The presented modification of the histopathological protocol is cheap, reproducible, and allows for a comprehensive evaluation of PCL scaffolds while maintaining their trabecular, honeycomb-like structure on cross-sections.

In Vivo Supportive Effects of Mesenchymal Stem Cells on Fat Graft Stabilization and Local Induction of Angiogenesis Are Not Dependent on the Cell Donor Age or In Vitro Cell Culture Duration.

Mesenchymal stromal cells from adipose tissue (adipose stromal cells, ASCs) are regulators of repair processes in situ by paracrine mechanisms. These unique capabilities make ASCs candidates for the regenerative medicine applications, including cell-assisted lipotransfer method. ASC aging processes have been extensively researched in vitro, there is however limited information about the impact of ASC aging on their biological role in tissue regeneration in vivo. The aim of our study was the research of the possible effects of aging processes of ASCs resulting from the donor age or from in vitro aging during long-term culture (ASC expansion in bioreactors) on their capability to support survival of adipose subcutaneous transplants in rats. The supportive in vivo effects of ASCs from young donors were compared with the effects of ASCs from old donors and ASCs "aged" in long-term in vitro cultures. Fat grafts enriched with ASCs (regardless of their age) retain their volume longer than fat grafts without ASCs supplementation. Vascular expansion in cell-enriched fat grafts was more intense when compared with the controls. It may be concluded that the aging of ASCs does not substantially reduce their ability for the support of the survival of adipose tissue grafts.

Comparison of the Donor Age-Dependent and In Vitro Culture-Dependent Mesenchymal Stem Cell Aging in Rat Model.

Clinical experiments suggest that mesenchymal stem cells (MSCs) may be useful for tissue repair therapies or treatment of the autoimmune disorders. There is still lack of consensus concerning the age limit of MSC donors, majority of researchers suggest the autologous MSC therapies of patients not exceeding age limit of 55-60 yrs. The purpose of our study was to compare the selected parameters of MSCs from adipose tissue (adipose stem cell, ASC) collected from young and old rats of ages corresponding to patient's ages 25 yrs. and 80 yrs., respectively. The differences of parameters of ASCs from young and old animals were compared with the differences between ASCs from short-term (3 passage) and long-term (30 passage) in vitro culture. Cell morphology, surface marker expression, growth potential, metabolic activity, ß-galactosidase activity, clonogenic potential, angiogenic potential, and differentiation ability of ASCs from young and aged animals and from in vitro cultures at 3rd and 30th passages were compared and analyzed. It may be concluded that ASCs may be applied for autologous transplantations in aged patients. Comparison of ASC aging dynamics depending on host aging or in vitro culture duration suggests that long-term in vitro culture may affect ASCs more than natural aging process of their host. We suggest that ASCs expanded in vitro prior to their clinical use must be carefully screened for the possible aging effects resulting not only from donor age, but from the duration of their in vitro culture.

Bioactive Nanofiber-Based Conduits in a Peripheral Nerve Gap Management-An Animal Model Study.

The aim was to examine the efficiency of a scaffold made of poly (L-lactic acid)-co-poly(ϵ-caprolactone), collagen (COL), polyaniline (PANI), and enriched with adipose-derived stem cells (ASCs) as a nerve conduit in a rat model. P(LLA-CL)-COL-PANI scaffold was optimized and electrospun into a tubular-shaped structure. Adipose tissue from 10 Lewis rats was harvested for ASCs culture. A total of 28 inbred male Lewis rats underwent sciatic nerve transection and excision of a 10 mm nerve trunk fragment. In Group A, the nerve gap remained untouched; in Group B, an excised trunk was used as an autograft; in Group C, nerve stumps were secured with P(LLA-CL)-COL-PANI conduit; in Group D, P(LLA-CL)-COL-PANI conduit was enriched with ASCs. After 6 months of observation, rats were sacrificed. Gastrocnemius muscles and sciatic nerves were harvested for weight, histology analysis, and nerve fiber count analyses. Group A showed advanced atrophy of the muscle, and each intervention (B, C, D) prevented muscle mass decrease (p < 0.0001); however, ASCs addition decreased efficiency vs. autograft (p < 0.05). Nerve fiber count revealed a superior effect in the nerve fiber density observed in the groups with the use of conduit (D vs. B p < 0.0001, C vs. B p < 0.001). P(LLA-CL)-COL-PANI conduits with ASCs showed promising results in managing nerve gap by decreasing muscle atrophy.

Cerebral Toxoplasmosis, CMV and Bacterial Pneumonia with Decreasing CD4+ T-Cell Count as Results of Antiretroviral Therapy Discontinuation-A Case Report.

Cerebral toxoplasmosis occurs mainly in immunocompromised hosts as a reactivation of latent Toxoplasma gondii infection. In the diagnostic process, magnetic resonance imaging (MRI), serum testing, and biopsy are used. We describe a case of a 43-year-old HIV-positive patient presenting with altered levels of consciousness, aphasia, and hemiparesis. The patient had a history of antiretroviral therapy discontinuation for about 3 years. MRI revealed lesions, suggesting cerebral toxoplasmosis and subacute hemorrhage, serum tests for Toxoplasma gondii were positive. Antiparasitics and glycocorticosteroids were administered. A decline in viral load and clinical improvement were observed, however CD4+ T-cell count continued to decrease. The patient's state worsened, he developed CMV and bacterial pneumonia, which led to his death. What is crucial in the management of an HIV-infected patient is effective and continuous antiretroviral therapy. Discontinuation of the treatment may result in AIDS and lead to poor recovery of the CD4+ T-cell population, even after reimplementation of antiretroviral therapy and a decrease in viral load.

The Effect of L-Ascorbic Acid and Serum Reduction on Tenogenic Differentiation of Human Mesenchymal Stromal Cells.

BACKGROUND AND OBJECTIVES: Despite significant improvement in the treatment of tendon injuries, the full tissue recovery is often not possible because of its limited ability to auto-repair. The transplantation of mesenchymal stromal cells (MSCs) is considered as a novel approach in the treatment of tendinopathies. The question about the optimal culture conditions remains open. In this study we aimed to investigate if serum reduction, L-ascorbic acid supplementation or a combination of both factors can induce tenogenic differentiation of human adipose-derived MSCs (ASCs). METHODS AND RESULTS: Human ASCs from 3 healthy donors were used in the study. The tested conditions were: 0.5 mM of ascorbic acid 2-phosphate (AA-2P), reduced serum content (2% FBS) or combination of these two factors. The combination of AA-2P and 2% FBS was the only experimental condition that caused a significant increase of the expression of all analyzed genes related to tenogenesis (SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3, DECORIN) in comparison to the untreated control (evaluated by RT-PCR, 5th day of experiment). Moreover, this treatment significantly increased the synthesis of SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3 proteins at the same time point (evaluated by Western blot method). Double immunocytochemical staining revealed that AA-2P significantly increased the extracellular deposition of both types of collagens. Semi-quantitative Electron Spin Resonance analysis of ascorbyl free radical revealed that AA-2P do not induce harmful transition metals-driven redox reactions in cell culture media. CONCLUSIONS: Obtained results justify the use of reduced content of serum with the addition of 0.5 mM of AA-2P in tenogenic inducing media.

Beneficial Effect of IL-4 and SDF-1 on Myogenic Potential of Mouse and Human Adipose Tissue-Derived Stromal Cells.

Under physiological conditions skeletal muscle regeneration depends on the satellite cells. After injury these cells become activated, proliferate, and differentiate into myofibers reconstructing damaged tissue. Under pathological conditions satellite cells are not sufficient to support regeneration. For this reason, other cells are sought to be used in cell therapies, and different factors are tested as a tool to improve the regenerative potential of such cells. Many studies are conducted using animal cells, omitting the necessity to learn about human cells and compare them to animal ones. Here, we analyze and compare the impact of IL-4 and SDF-1, factors chosen by us on the basis of their ability to support myogenic differentiation and cell migration, at mouse and human adipose tissue-derived stromal cells (ADSCs). Importantly, we documented that mouse and human ADSCs differ in certain reactions to IL-4 and SDF-1. In general, the selected factors impacted transcriptome of ADSCs and improved migration and fusion ability of cells in vitro. In vivo, after transplantation into injured muscles, mouse ADSCs more eagerly participated in new myofiber formation than the human ones. However, regardless of the origin, ADSCs alleviated immune response and supported muscle reconstruction, and cytokine treatment enhanced these effects. Thus, we documented that the presence of ADSCs improves skeletal muscle regeneration and this influence could be increased by cell pretreatment with IL-4 and SDF-1.

Copper Does Not Induce Tenogenic Differentiation but Promotes Migration and Increases Lysyl Oxidase Activity in Adipose-Derived Mesenchymal Stromal Cells.

BACKGROUND: Copper belongs to the essential trace metals that play a key role in the course of cellular processes maintaining the whole body's homeostasis. As there is a growing interest in transplanting mesenchymal stromal cells (MSCs) into the site of injury to improve the regeneration of damaged tendons, the purpose of the study was to verify whether copper supplementation may have a positive effect on the properties of human adipose tissue-derived MSCs (hASCs) which potentially can contribute to improvement of tendon healing. RESULTS: Cellular respiration of hASCs decreased with increasing cupric sulfate concentrations after 5 days of incubation. The treatment with CuSO4 did not positively affect the expression of genes associated with tenogenesis (COL1α1, COL3α1, MKX, and SCX). However, the level of COL1α1 protein, whose transcript was decreased in comparison to a control, was elevated after a 5-day exposition to 25 µM CuSO4. The content of the MKX and SCX protein in hASCs exposed to cupric sulfate was reduced compared to that of untreated control cells, and the level of the COL3α1 protein, whose transcript was decreased in comparison to a control, was elevated after a 5-day exposition to 25 µM CuSO4. The content of the MKX and SCX protein in hASCs exposed to cupric sulfate was reduced compared to that of untreated control cells, and the level of the COL3. CONCLUSION: Copper sulfate supplementation can have a beneficial effect on tendon regeneration not by inducing tenogenic differentiation, but by improving the recruitment of MSCs to the site of injury, where they can secrete growth factors, cytokines and chemokines, and prevent the effects of oxidative stress at the site of inflammation, as well as improve the stabilization of collagen fibers, thereby accelerating the process of tendon healing.

The Fluctuations of Leukocytes and Circulating Cytokines in Septic Humanized Mice Vary With Outcome.

Sepsis remains a major challenge in translational research given its heterogeneous pathophysiology and the lack of specific therapeutics. The use of humanized mouse chimeras with transplanted human hematopoietic cells may improve the clinical relevance of pre-clinical studies. However, knowledge of the human immuno-inflammatory response during sepsis in humanized mice is scarce; it is unclear how similar or divergent mouse and human-origin immuno-inflammatory responses in sepsis are. In this study, we evaluated the early outcome-dependent immuno-inflammatory response in humanized mice generated in the NSG strain after cecal ligation and puncture (CLP) sepsis. Mice were observed for 32 h post-CLP and were assigned to either predicted-to-die (P-DIE) or predicted-to-survive (P-SUR) groups for retrospective comparisons. Blood samples were collected at baseline, 6 and 24 h, whereas the bone marrow and spleen were collected between 24 and 32 h post-CLP. In comparison to P-SUR, P-DIE humanized mice had a 3-fold higher frequency of human splenic monocytes and their CD80 expression was reduced by 1.3-fold; there was no difference in the HLA-DR expression. Similarly, the expression of CD80 on the bone marrow monocytes from P-DIE mice was decreased by 32% (p < 0.05). Sepsis induced a generalized up-regulation of both human and murine plasma cytokines (TNFα, IL-6, IL-10, IL-8/KC, MCP-1); it was additionally aggravated in P-DIE vs. P-SUR. Human cytokines were strongly overridden by the murine ones (approx. ratio 1:9) but human TNFα was 7-fold higher than mouse TNFα. Interestingly, transplantation of human cells did not influence murine cytokine response in NSG mice, but humanized NSG mice were more susceptible to sepsis in comparison with NSG mice (79 vs. 33% mortality; p < 0.05). In conclusion, our results show that humanized mice reflect selected aspects of human immune responses in sepsis and therefore may be a feasible alternative in preclinical immunotherapy modeling.

Safety of adipose-derived cell (stromal vascular fraction - SVF) augmentation for surgical breast reconstruction in cancer patients.

BACKGROUND: Progress in breast cancer surgery results in a decreased frequency of mastectomy, in the early phases of cancer replaced by breast conserving therapy (lumpectomy). Increased popularity of breast reconstruction by fat or adipose stem cells (ASC)-enriched fat transfer raised uncertainty about the possible risk of increased cancer recurrence. In vitro studies suggest that locally secreted cytokines and reconstructed local blood vessels may stimulate cancer expansion or cancer de novo induction from glandular tissue remaining after lumpectomy. OBJECTIVES: The purpose of the study was to evaluate the risk of cancer recurrence in breast cancer patients related to the stromal vascular fraction (SVF) augmentation during autologous fat grafting for breast reconstruction. MATERIAL AND METHODS: The tumor recurrence ratio in 56 patients having the breast reconstructed with autologous ASC (transplanted as the subpopulation present in SVF) was compared with the frequency of tumor recurrence in 252 matched patients treated in clinics without subsequent breast reconstruction. Adipose tissue was collected by the Coleman technique and split into 2 portions: one was used for breast reconstruction, the other was enzymatically digested, and isolated cells were used for the augmentation of fat implanted into the breast area. Cancer recurrence in the experimental and matched control group was evaluated following 3-year-long observation time, and the statistical significance of difference in cancer recurrence between the experimental and control group was evaluated. RESULTS: Cancer recurrence in the group of patients treated with ASC-enriched fat for breast reconstruction was 3.7% and did not differ significantly from the control group data (4.13%). No adverse effects of therapy were observed. CONCLUSIONS: Our study does not produce any data suggesting increased cancer risk following breast reconstruction after a mastectomy or a lumpectomy combined with local radiotherapy. It may be concluded that an autologous transplantation of fat augmented with ASC is a safe and efficient procedure. Longer observation time and the observation of larger numbers of patients would be useful for strengthening the conclusion.

Bmp-12 activates tenogenic pathway in human adipose stem cells and affects their immunomodulatory and secretory properties.

BACKGROUND: Cell-based therapy is a treatment method in tendon injuries. Bone morphogenic protein 12 (BMP-12) possesses tenogenic activity and was proposed as a differentiating factor for stem cells directed to transplantation. However, BMPs belong to pleiotropic TGF-ß superfamily and have diverse effect on cells. Therefore, the aim of this study was to determine if BMP-12 induces tenogenic differentiation of human adipose stem cells (hASCs) and how it affects other features of this population. RESULTS: Human ASCs from 6 healthy donors were treated or not with BMP-12 (50 or 100 ng/ml, 7 days) and tested for gene expression (COLL1, SCX, MKH, DCN, TNC, RUNX2), protein expression (COLL1, COLL3, MKH), proliferation, migration, secretory activity, immunomodulatory properties and susceptibility to oxidative stress. RT-PCR revealed up-regulation of SCX, MKH and RUNX2 genes in BMP-12 treated cells (2.05, 2.65 and 1.87 fold in comparison to control, respectively, p < 0.05) and Western Blot revealed significant increase of COLL1 and MHK expression after BMP-12 treatment. Addition of BMP-12 significantly enhanced secretion of VEGF, IL-6, MMP-1 and MPP-8 by hASCs while had no effect on TGF-ß, IL-10, EGF and MMP-13. Moreover, BMP-12 presence in medium attenuated inhibitory effect of hASCs on allo-activated lymphocytes proliferation. At the same time BMP-12 displayed no influence on hASCs proliferation, migration and susceptibility to oxidative stress. CONCLUSION: BMP-12 activates tenogenic pathway in hASCs but also affects secretory activity and impairs immunomodulatory potential of this population that can influence the clinical outcome after cell transplantation.

Clinical Application of Autologous Adipose Stem Cells in Patients with Multiple Sclerosis: Preliminary Results.

The clinical outcome of autologous adipose stem cell (ASC) treatment of patients with multiple sclerosis (MS) was investigated following one year of observation. Methods. The clinical and MRI outcomes of 16 ASC-treated patients with RRMS and SPMS are reported after a one-year follow-up period. Results. At 18 months of follow-up, some patients showed "enticing" improvements on some exploratory efficacy measures, although a significant benefit was not observed for any measure across the entire group. Neither the progression of disability nor relapses were observed in any cases. In four patients, we found new gadolinium+ (Gd+) lesions on MRI. Our results indicate that ASC therapy is safe and does not produce any substantial side effects. Disease progression-free survival (PFS) of 18 months was seen in all patients with RRMS and SPMS. In these patients, EDSS scores did not progress above baseline scores. Gd-enhancing lesions were observed in two cases with RRMS, but these patients did not exhibit changes in EDSS score. Conclusion. Intrathecal treatment with ASCs is an attractive form of therapy for patients with MS but should be reserved for cases with aggressive disease progression, for cases that are still in the inflammatory phase, and for the malignant form.

Adipose-Derived Cells (Stromal Vascular Fraction) Transplanted for Orthopedical or Neurological Purposes: Are They Safe Enough?

Although mesenchymal stem cells are used in numerous clinical trials, the safety of their application is still a matter of concern. We have analysed the clinical results of the autologous adipose-derived stem cell treatment (stromal vascular fraction (SVF) containing adipose-derived stem cells, endothelial progenitors, and blood mononuclear cells) for orthopedic (cartilage, bone, tendon, or combined joint injuries) and neurologic (multiple sclerosis) diseases. Methods of adipose tissue collection, cell isolation and purification, and resulting cell numbers, viability, and morphology were considered, and patient's age, sex, disease type, and method of cell administration (cell numbers per single application, treatment numbers and frequency, and methods of cell implantation) were analysed and searched for the unwanted clinical effects. Results of cellular therapy were compared retrospectively to those obtained with conventional medication without SVF application. SVF transplantation was always the accessory treatment of patients receiving "standard routine" therapies of their diseases. Clinical experiments were approved by the Bioethical Medical Committees supervising the centers where patients were hospitalised. The conclusion of the study is that none of the treated patients developed any serious adverse event, and autologous mesenchymal stem (stromal) cell clinical application is a safe procedure resulting in some beneficial clinical effects (not analysed in this study).

Ultrastructure of Candida albicans pleomorphic forms: phase-contrast microscopy, scanning and transmission electron microscopy.

A modified method of glutaraldeyde-osmium tetroxide fixation was adjusted to characterize the ultrastructure of Candida albicans pleomorphic forms, using phase-contrast microscopy, scanning electron microscopy and transmission electron microscopy. The discovered morphological criteria defining the individual morphotypes are discussed in terms of mycological and histopathological diagnostics of candidiasis. The relations are discussed between fungal pleomorphism, virulence and susceptibility of different morphotypes to fungicides.

[Virulence factors of Candida albicans]. / Czynniki zjadliwosci Candida albicans.

Candida albicans is the most common etiological factor of opportunistic human fungal infections. In this review, we focus on the major virulence factors that mediate the pathogenesis of C. albicans. Among these virulence factors, secreted aspartyl proteases, adherence, pleomorphism are the most important features of C. albicans infections. Ability to exist as different pleomorphic forms is defined as pleomorphism. A number of quorum sensing (QS) molecules have been described which affect morphogenesis process in C. albicans. Furthermore, the morphological transition of C. albicans in response to changing environmental conditions represent a means by which the strain adapts to different biological niches. Furthermore, every morphotype has own virulence profile and each pleomorphic form provide critical functions required for pathogenesis. Candida albicans is a producer of extracellular hydrolytic enzymes. Among them lipases, phospholipases and secreted aspartyl proteinases (Sap) are most significant in virulence. Sap proteins contribute to pathogenesis by digestion of host cell membranes and molecules of the host immune system to avoid antimicrobial attack by the host. One of the key features in the development of candidiasis is adhesion ofC. albicans to buccal and vaginal epithelial cells. The adhesion to host cells represents the first step in the internalization process which involves adhesins. Knowledge of the role of the various C. albicans' virulence factors during in vivo infections is still incomplete, therefore further studies including quantification of genes expression and histopathological examination of tissues damage are required to fully understand pathogenesis of this opportunistic pathogen.

In vitro study of secreted aspartyl proteinases Sap1 to Sap3 and Sap4 to Sap6 expression in Candida albicans pleomorphic forms.

Transition from round budding cells to long hyphal forms and production of secreted aspartic proteases (Saps) are considered virulence-associated factors of Candida albicans. Although plenty of data dealing with Saps involvement in the infection process have been published, Saps expression by the different pleomorphic forms as well as the capacity of C. albicans filaments to express Sap1-6 under serum influence are poorly investigated. In this study, we used immunofluorescence and immunoelectron microscopy for the detection of Sap1-6 isoenzymes in C. albicans pleomorphic cells (blastoconidia, germ tubes, pseudohyphae, true hyphae) grown in Sap-inductive human serum and Sap non-inductive medium - yeast extract-peptone-glucose (YEPD). Isoenzymes were below the detection level in all blastoconidial cells grown in YEPD for 18 h. Sap1-6 expression was hardly detected in C. albicans cells cultivated in serum for 20 min. Increasing level of Sap1-6 expression was observed when C. albicans was incubated for 2, 6 and 18 h in serum corresponding to the development of germ tubes, pseudohyphae and true hyphae. The expression of Sap1-3 in pseudohyphae and true hyphae was more intensive compared to Sap4-6. Thus, we could show that human serum induced hyphae formation and the expression of Sap1-6 were co-regulated.