The immunogenicity of p24 protein from HIV-1 virus is strongly supported and modulated by coupling with liposomes and mannan.

Anti-viral and anti-tumor vaccines aim to induce cytotoxic CD8+ T cells (CTL) and antibodies. Conserved protein antigens, such as p24 from human immunodeficiency virus, represent promising component for elicitation CTLs, nevertheless with suboptimal immunogenicity, if formulated as recombinant protein. To enhance immunogenicity and CTL response, recombinant proteins may be targeted to dendritic cells (DC) for cross presentation on MHCI, where mannose receptor and/or other lectin receptors could play an important role. Here, we constructed liposomal carrier-based vaccine composed of recombinant p24 antigen bound by metallochelating linkage onto surface of nanoliposomes with surface mannans coupled by aminooxy ligation. Generated mannosylated proteonanoliposomes were analyzed by dynamic light scattering, isothermal titration, and electron microscopy. Using murine DC line MutuDC and murine bone marrow derived DC (BMDC) we evaluated their immunogenicity and immunomodulatory activity. We show that p24 mannosylated proteonanoliposomes activate DC for enhanced MHCI, MHCII and CD40, CD80, and CD86 surface expression both on MutuDC and BMDC. p24 mannosylated liposomes were internalized by MutuDC with p24 intracellular localization within 1 to 3 h. The combination of metallochelating and aminooxy ligation could be used simultaneously to generate nanoliposomal adjuvanted recombinant protein-based vaccines versatile for combination of recombinant antigens relevant for antibody and CTL elicitation.

Diagnostic value of serum galactomannan, (1,3)-ß-d-glucan, and Aspergillus fumigatus-specific IgA and IgG assays for invasive pulmonary aspergillosis in non-neutropenic patients.

Detection of serum galactomannan (GM) and (1,3)-ß-d-glucan (BG) is considered useful for non-culture diagnosis of invasive pulmonary aspergillosis (IPA) in neutropenic patients. Only few studies evaluated these seromarkers in non-neutropenic patients suspected of having IPA. The aim of this study was to evaluate both tests together with the Aspergillus fumigatus-specific serum IgG and IgA (IgAG) test for serological IPA diagnosis in non-neutropenic patients. Sera from 87 patients suspected of having IPA were retrospectively analysed. Patients were categorised into groups of proven IPA (n = 10), putative IPA (n = 31) and non-IPA colonisation (n = 46). When the GM, BG and IgAG assays were used for patients included in the study, the sensitivity/specificity/positive predictive value (PPV)/negative predictive value (NPV) were 48.8%/91.3%/83.3%/66.7%, 82.9%/73.9%/73.9%/82.9% and 75.6%/95.7%/93.9%/81.5%, respectively. Thus, the highest specificity and PPV were confirmed for the IgAG assay. Improvements in the sensitivity and NPV were achieved by "at least one positive" analysis with the GM and BG assays, with the sensitivity/specificity/PPV/NPV values being 85.0%/69.6%/71.4%/84.2%. Nevertheless, the highest sensitivity and NPV were achieved by the "at least one positive" analysis combining the GM, BG and IgAG tests (97.6% and 96.8%, respectively). The involvement of the IgAG assay could improve IPA diagnosis in non-neutropenic patients by increasing the sensitivity and NPV when combined with the GM or BG assays. Furthermore, improvement was achieved by combining the GM, BG and IgAG assays using the "at least one positive test" strategy, especially if doubt exists.

Patients with IgA nephropathy have altered levels of immunomodulatory C19 steroids. Glucocorticoid therapy with addition of adrenal androgens may be the choice.

Glucocorticoid (GC) therapy is one of the methods of choices for treatment of autoimmune diseases (ADs). In addition, adrenal androgens are known as immunoprotective GC-antagonists. Adrenal steroids preferentially influence the Th1-components over the Th2 ones. We investigated steroid metabolome (using gas chromatography-mass spectrometry) in healthy controls (H), GC-untreated patients with ADs different from IgA nephropathy (U), GC-treated patients with ADs different from IgA nephropathy (T) and in patients with IgA nephropathy (IgAN), which were monitored on the beginning (N0), after one week (N1) and after one month (N2) of prednisolone therapy (60 mg of prednisolone/day/m(2) of body surface). Between-group differences were assessed by one-way ANOVA, while the changes during the therapy were evaluated by repeated measures ANOVA. The ANOVA testing was followed by Duncan's multiple comparisons. IgAN patients and patients with other ADs exhibited lack of adrenal androgens due to attenuated activity of adrenal zona reticularis (ZR). Androgen levels including their 7alpha-, 7beta-, and 16alpha-hydroxy-metabolites were further restrained by GC-therapy. Based on these results and data from the literature, we addressed the question, whether a combination of GCs with delta(5)-steroids or their more stable synthetic derivatives may be optimal for the treatment of antibodies-mediated ADs.

Endotoxin-minimized HIV-1 p24 fused to murine hsp70 activates dendritic cells, facilitates endocytosis and p24-specific Th1 response in mice.

Heat shock proteins hsp70 and gp96 have been confirmed as adjuvants enabling induction of cell- and antibody-mediated immunity specific to associated protein or peptide antigens due to the activation of naive dendritic cells and supporting cross-presentation of associated antigen. An efficacious vaccine preventing HIV-1 infection should induce (1) antibodies neutralizing HIV-1 Env protein, preventing virus spreading and (2) CD4(+) Th1 and CD8(+) T cells specific to viral proteins, especially gag p24, important for elimination of HIV-1 infected cells. As p24 is relatively poorly recognized by dendritic cells, its targeting to DC is important for enhancement of vaccine efficacy. In this study, a p24 protein fused to the C- or N-terminus of murine hsp70 was produced as a recombinant protein and administered without any adjuvant to experimental BALB/c mice. Consequently, p24-specific cellular and humoral immune responses were measured. To minimize the effect of bacterial endotoxin, each protein was subjected to a repeated endotoxin phase extraction until each preparation contained less than 2.5 endotoxin unit (EU) per mg of antigen. In addition, endocytosis of p24 fused to hsp70 by dendritic cells and their activation were characterized. The fusion to hsp70 protein enhanced endocytosis of p24 as well as activation of dendritic cells in vitro. After immunization of mice, hsp70-p24 fusion protein induced the strongest p24-specific CD4(+) and CD8(+) T cells (IFN-γ production) and humoral (IgG2b) responses corresponding to Th1 type dominance, whereas p24-hsp70 or p24 itself induced weaker responses.

Bone remodeling, particle disease and individual susceptibility to periprosthetic osteolysis.

Bone remodeling is a tightly coupled process consisting of repetitive cycles of bone resorption and formation. Both processes are governed by mechanical signals, which operate in conjunction with local and systemic factors in a discrete anatomic structure designated a basic multicellular unit (BMU). The microenvironment around total joint arthroplasty is a dynamic and complex milieu influenced by the chemical and physical stimuli associated with servicing the prosthesis. A key factor limiting the longevity of the prosthesis is polyethylene wear, which induces particle disease, and this may lead to increased and prolonged activity of BMUs resulting in periprosthetic osteolysis. Several pathways regulating BMU function have been reported in the past, including RANKL/RANK/OPG/TRAF6, TNF-alpha/TNFR/TRAF1, and IL-6/CD126/JAK/STAT. Moreover, the expression and functional activity of all these molecules can be affected by variations in their genes. These may explain the differences in severity of bone defects or prosthetic failure between patients with similar wear rates and the same prosthesis. Simultaneously, this data strongly support the theory of individual susceptibility to prosthetic failure.

Candidiasis--do we need to fight or to tolerate the Candida fungus?

Candidiases, infections caused by germination forms of the Candida fungus, represent a heterogeneous group of diseases from systemic infection, through mucocutaneous form, to vulvovaginal form. Although caused by one organism, each form is controlled by distinct host immune mechanisms. Phagocytosis by polymorphonuclears and macrophages is generally accepted as the host immune mechanism for Candida elimination. Phagocytes require proinflammatory cytokine stimulation which could be harmful and must be regulated during the course of infection by the activity of CD8+ and CD4+ T cells. In the vaginal tissue the phagocytes are inefficient and inflammation is generally an unwanted reaction because it could damage mucosal tissue and break the tolerance to common vagina antigens including the otherwise saprophyting Candida yeast. Recurrent form of vulvovaginal candidiasis is probably associated with breaking of such tolerance. Beside the phagocytosis, specific antibodies, complement, and mucosal epithelial cell comprise Candida eliminating immune mechanisms. They are regulated by CD4+ and CD8+ T cells which produce cytokines IL-12, IFN-gamma, IL-10, TGF-beta, etc. as the response to signals from dendritic cells specialized to sense actual Candida morphotypes. During the course of Candida infection proinflammatory signals (if initially necessary) are replaced successively by antiinflammatory signals. This balance is absolutely distinct during each candidiasis form and it is crucial to describe and understand the basic principles before designing new therapeutic and/or preventive approaches.

Comparison of protective effect of protein and DNA vaccines hsp90 in murine model of systemic candidiasis.

Preventive vaccination by a hsp90-expressing DNA vaccine and recombinant hsp90 protein vaccine, both derived from the Candida albicans hsp90 using BALB-c mouse model of systemic candidiasis, was performed. Hsp90 mRNA was cloned from a clinical isolate of C. albicans, converted to cDNA and cloned into vaccination plasmid pVAX1. Two methods of DNA application were tested: intramuscular (i.m.) and intradermal (i.d.) injection. Recombinant protein was applied by i.d. injection with Freund's adjuvant; the control groups received PBS or Freund's adjuvant only. Mice were vaccinated and after 19 d re-vaccinated. After 3 weeks, the mice were challenged with the live C. albicans in a dose of 5 x 10(6) CFU per mouse. After the challenge, the mice vaccinated i.d. with DNA vaccine survived for 39 and 64% longer compared to those receiving Freund's adjuvant and/or PBS, respectively. The i.m. application of the DNA vaccine did not provide any significant protectivity. The serum level of anti-candida-hsp90 serum IgG antibodies correlated with the survival rate in both i.d. protein and DNA vaccination approaches. We stressed the importance of specific humoral immunity in the mouse model of systemic candidiasis.

Isolation and characterization of an immunogenic fragment of heat shock protein 60 from Trichophyton mentagrophytes.

Heat shock protein 60 (hsp60) were isolated from several fungal, protozoal and many bacterial pathogens and successfully used for protective vaccination in some infection models. This work focuses on the isolation of recombinant hsp60 from the dermatophyte, Trichophyton mentagrophytes as a potentially protective antigen in trichophytosis. With the help of a previously tested set of degenerated primers, it was used reverse transcriptase polymerase chain reaction (RT-PCR) for isolation of partial cDNA of the hsp60 T. mentagrophytes (labelled hsp60-TM814), which was cloned into cloning vector. The sequencing of hsp60-TM814 cDNA and global alignment confirmed homology of the hsp60-TM814 with other members of the hsp60 family. Hsp60-TM814 cDNA corresponds to the region encoding the immunoprotective fragment of the hsp60 from Histoplasma capsulatum, used successfully in mouse model of histoplasmosis. A recombinant fragment (r-hsp60-TM664), 220 amino acids in length, was prepared in a prokaryote expression system, and its identity confirmed by mass spectroscopy. High immunogenicity of r-hsp60-TM664 was proven after subcutaneous immunization of mice. Immunized mouse sera recognized r-hsp60-TM664 on Western blots as well as hsp60 from mouse liver lysate and lysate of Candida albicans.

Allergen standardization.

The article summarises current knowledge about the preparation and standardisation of treatment and diagnosis of allergies. It summarises the basic conceptions, units, methods and conditions for preparation, conservation and the storage of allergens. The article is especially orientated towards grass pollen.

Development of the primer set for the detection of the hsp60 gene in Trichophyton mentagrophytes cDNA.

Three sequences of hsp60 from Saccharomyces cerevisiae, Schizosaccharomyces pombe and Histoplasma capsulatum were compared. Local multiple alignment of these sequences allowed the selection of two oligonucleotides suitable as primers for the polymerase chain reaction. This primer set was used for the amplification of a part of the hsp60 gene from cDNA of Trichophyton mentagrophytes and S. cerevisiae. Similar fragments detected in both PCR's imply the possible future use of the developed primer set for the detection of the hsp60 gene in other fungal species.

Heat shock proteins in immune reactions.

The review concerns heat shock proteins and their significance in immune reactions. It focuses on problems of physiological and pathological interactions in etiology and duration of autoimmune diseases and infection processes, especially fungal infections. New trends are described in exploitation of heat shock proteins for preparation of specific protective vaccines.

Electrophoretic patterns of Trichophyton mentagrophytes mutants with changed growth rate at different cultivation temperatures.

Five mutants of Trichophyton mentagrophytes with changed optimum cultivation temperature and the wild strain were used in this study. The growth rates, the keratinolytic activities and the 1-D electrophoretic patterns of these mutants were examined repeatedly. The differences among the individual mutants and the wild strain can be useful for a better understanding of the participating individual proteins for thermotolerance of this fungus.

Detection of antigens in mycelial and in arthroconidial phases of Trichophyton mentagrophytes.

Protein pattern changes were investigated in the filamentous fungus Trichophyton mentagrophytes during the morphological transition induced by increased temperature and higher CO2 partial pressure in cultivation atmosphere. The differences between the mycelial and the arthroconidial phase were characterized by SDS-PAGE and by immunodetection with mouse polyclonal antibodies. The components found by Western blotting in mycelia were 88, 86, 32, 29, 19.5, 18.5 kDa, in arthroconidia 108, 92, 88, 66, 56, 41, 39, 19.5 kDa. The results suggest the participation of some heat shock associated proteins of T. mentagrophytes in host immune response against mycotic infection.