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Dendritic Cells Pulsed with Fungal RNA Induce Protective Immunity to Candida albicans in Hematopoietic Transplantation
Angela Bacci, Claudia Montagnoli, Katia Perruccio, Silvia Bozza, Roberta Gaziano, Lucia Pitzurra, Andrea Velardi, Cristiana Fe' d'Ostiani, Jim E. Cutler and Luigina Romani
J Immunol March 15, 2002, 168 (6) 2904-2913; DOI: https://doi.org/10.4049/jimmunol.168.6.2904
Angela Bacci
Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, and
Claudia Montagnoli
Katia Perruccio
Division of Hematology, Clinical Immunology, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy;
Silvia Bozza
Roberta Gaziano
Microbiology Section, Department of Experimental Medicine, University of Rome, Tor Vergata, Rome, Italy; and
Lucia Pitzurra
Andrea Velardi
Cristiana Fe' d'Ostiani
Jim E. Cutler
Department of Microbiology, Montana State University, Bozeman, MT 59717
Luigina Romani
Immature myeloid dendritic cells (DC) phagocytose yeasts and hyphae of the fungus Candida albicans and induce different Th cell responses to the fungus. Ingestion of yeasts activates DC for production of IL-12 and Th1 priming, while ingestion of hyphae induces IL-4 production and Th2 priming. In vivo, generation of antifungal protective immunity is induced upon injection of DC ex vivo pulsed with Candida yeasts but not hyphae. In the present study we sought to determine the functional activity of DC transfected with yeast or hyphal RNA. It was found that DC, from either spleens or bone marrow, transfected with yeast, but not hyphal, RNA 1) express fungal mannoproteins on their surface; 2) undergo functional maturation, as revealed by the up-regulated expression of MHC class II Ags and costimulatory molecules; 3) produce IL-12 but no IL-4; 4) are capable of inducing Th1-dependent antifungal resistance when delivered s.c. in vivo in nontransplanted mice; and 5) provide protection against the fungus in allogeneic bone marrow-transplanted mice, by accelerating the functional recovery of Candida-specific IFN-γ-producing CD4+ donor lymphocytes. These results indicate the efficacy of DC pulsed with Candida yeasts or yeast RNA as fungal vaccines and point to the potential use of RNA-transfected DC as anti-infective vaccines in conditions that negate the use of attenuated microorganisms or in the case of poor availability of protective Ags.
In humans, Candida albicans is the most frequently isolated fungal pathogen (1). The delicate balance between the host and this otherwise harmless commensal fungus turn into a parasitic relationship, resulting in the development of severe infections. Invasive candidiasis is a significant cause of morbidity and mortality in differently immunocompromised patients, including those undergoing bone marrow transplantation (BMT).3 It is recognized that the nature and extent of the impairment of host defense influence the manifestation and severity of infection (1).
Clinical evidence and experimental data indicate that both the innate and the adaptive immune systems regulate resistance to Candida infections (3, 4, 5). In murine experimental models of infection, it has been demonstrated that Th cell reactivity plays a central role in regulating immune responses to the fungus, Th1 reactivity being responsible for resistance and Th2 reactivity being associated with susceptibility (6). The development of protective anticandidal Th1 responses requires the concerted actions of several cytokines, including IFN-γ and IL-12, in the relative absence of Th2 cytokines, such as IL-4 and IL-10, which inhibit development of Th1 responses (6).
However, the fungus is not a mere passive participant in the infectious process, and a hypothetical set of virulence factors has been attributed to it (7). Among these, the ability to reversibly switch between budding yeast to the filamentous growth form or hypha, all of which can be found in infected tissues (1), is thought to be important for virulence (8, 9, 10).
Recent evidence indicates that dendritic cells (DC) are uniquely capable of decoding the fungus-associated information required to elicit the qualitative nature of the adaptive immune response (11). DC finely discriminated between the two forms of C. albicans in terms of type of immune responses elicited. By the production of IL-12 and IL-4 in response to the nonvirulent and virulent forms of the fungus, DC were uniquely capable of Th priming and education in vitro and in vivo (11). This finding is particularly relevant in candidiasis, because the fungus behaves as a commensal as well as a true pathogen of skin and mucosal surfaces (1), known to be highly enriched for DC.
DC are uniquely specialized to initiate T cell immunity in vitro and in vivo (12). When delivered in vivo, DC charged with peptide Ags, tumor lysates, or viral vectors that encode the relevant Ags induce immune responses, which include protective and therapeutic immunity to tumors and pathogens in animals (11, 13, 14, 15, 16, 17, 18) and clinical antitumor responses in humans (19, 20, 21, 22).
Studies have shown that DC transfected with tumor RNA could also serve as potent cancer vaccines (23, 24, 25). DC transfected with chicken OVA RNA stimulate primary anti-OVA CTL responses in vitro. Moreover, vaccination of mice with OVA RNA-transfected DC has been used to treat OVA-positive tumor metastases to lung (23). These findings are particularly relevant, as they would extend vaccination to conditions of limited availability of protective Ags. However, whether or not induction of antimicrobial immunity with DC transfected with pathogen-derived RNA could represent a strategy of DC-based therapy in infections remains to be determined.
We have already shown that DC pulsed with viable Candida yeasts, but not hyphae, were capable of inducing protective Th1 immune responses when adoptively transferred in vivo (11). In the current study we tested whether transfecting DC with Candida yeast RNA is an effective way to induce antifungal protective immunity in vivo. Ex vivo transfected DC were adoptively transferred into recipient mice, and parameters of infection and immunity to it were evaluated upon the i.v. challenge with the fungus. We also used a very stringent experimental system to assess the priming capacity of transfected DC, which includes the infusion of DC in an allogeneic T-depleted BMT experimental model (26). In this model, autologous reconstitution of host stem cells is greatly reduced to the benefit of a long-term, donor-type chimerism in >95% of the mice and low incidence of graft-vs-host disease (27, 28, 29). We found here that DC transfected with yeast, but not hyphal, RNA were capable of stimulating Candida-specific Th1 immunity that conferred antifungal resistance to susceptible mice, either untransplanted or after allogeneic hematopoietic transplantation.
Female, 8- to 10-wk-old, inbred C3H/HeJ and hybrid (BALB/c × DBA/2)F1 (CD2F1) mice were obtained from Charles River Breeding Laboratories (Calco, Italy). Breeding pairs of homozygous IFN-γ-deficient (IFN-γ−/−) mice raised on BALB/c background (kindly provided by Dr. M. Kopf, Basel Institute for Immunology, Basel, Switzerland) were bred under specific pathogen-free conditions at the breeding facilities of the University of Perugia (Perugia, Italy). Mice of both sexes, 8–10 wk old, were used. Hybrid CD2F1 mice were used as wild-type mice. Bone marrow (BM)-transplanted mice were kept in small sterile cages (five animals in each cage) and fed with sterile food and water. Procedures involving animals and their care were conducted in conformity with national and international laws and policies.
Irradiation
C3H/HeJ mice were exposed to a single, lethal dose of 9 Gy from a gamma beam 150A, 60Co source (Clinac 600/C Varian; Cernusco, Milan, Italy) with focus to skin distance of 75 cm and 0.7 Gy/min dose rate. Unless BM-transplanted, mice die within 14 days.
C. albicans strains and isolation of fungal RNA
The origin and characteristics of C. albicans highly virulent and live vaccine strains used in this study have already been described (11). Total RNA was isolated from actively growing yeasts and hyphae as described (30). Briefly, cells were disrupted by repeated cycles of thawing and freezing on liquid nitrogen. Hot extraction buffer was added to the cells (a 1:1 mixture of phenol and 0.1 M LiCl, 100 mM Tris-HCl (pH 8), 10 mM EDTA, and 1% SDS at 80°C) and a mixture (24:1, v/v) of chloroform and isoamyl alcohol was then added to the cells. After centrifugation at 10,000 rpm at 4°C, the water phase was removed and mixed with an equal volume of 4 M LiCl. RNA was precipitated overnight at −20°C and collected by centrifugation at 10,000 rpm at 4°C. The RNA pellet was dissolved in water and precipitated with 3 M sodium acetate (pH 5.2) and ethanol at −20°C. Total RNA was quantitated by measuring OD at 260 and 280 nm. OD260/280 ratios ranged from 1.7 to 2. RNA was pelleted, washed with ethanol, air dried, and dissolved in water.
Preparation of T cell-depleted BM cells
Donor BM cells were prepared by differential agglutination with soybean agglutinin, as described (27). T cell-depleted soybean agglutinin-positive cells (containing <1% of contaminating T cells on FACS analysis) were injected at the concentration of ≥4 × 106/ml into recipient mice i.v. According to previous studies (27, 28, 29), >95% of the mice survived showing stable, donor-type hematopoietic chimerism, as revealed by donor-type MHC class I Ag expression on cells from spleens.
Purification, Candida pulsing, and culture of DC
DC were purified from spleens (SP-DC) by magnetic cell sorting with MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) conjugated to hamster anti-mouse CD11c mAbs (clone N-418), as described (11). Briefly, after overnight plastic adherence to remove macrophages, 108 collagenase D (Sigma-Aldrich, St. Louis, MO)-treated nonadherent spleen cells were reacted with 100 μl of CD11c MicroBeads before magnetic separation. BM-derived DC (BM-DC) were prepared as described (31). Briefly, BM-DC, obtained from femurs of mice, were depleted of red cells and seeded (2 × 107/ml) in six-well plates (Falcon; BD Labware, Lincoln Park, NJ) in 3 ml IMDM (Life Technologies, Milan, Italy) with 10% FCS, 50 mM 2-ME, 50 μg/ml gentamicin sulfate, 2000 U/ml GM-CSF (Sigma-Aldrich), and 1 × 103 U/ml IL-4 (BD PharMingen, San Diego, CA) for 6 days. On day 3 of culture, nonadherent cells were removed and fresh medium containing GM-CSF and IL-4 was added. On day 6 of culture, DC were isolated by transferring nonadherent cells to new culture plates and incubating at 37°C for at least 2 h. Consistent with previous reports (31, 32), >90% SP-DC (at ∼1% yield recovery) routinely express high levels of CD11c integrin, while BM-DC (at ∼10% of the BM population) uniformly showed low level of CD11c staining. More than 90% of BM-DC did not react with the 2.43 anti-CD8α mAb, while 25–30% of SP-DC expressed CD8α (data not shown). For pulsing, 2 × 106 DC were exposed to live yeasts or hyphae (at DC:yeast ratio of 1:1 and 1:10, respectively) for 2 h before addition of 2.5 μg/ml amphotericin B (Sigma-Aldrich) to prevent Candida overgrowth, as described (11). Pulsing of DC with RNA was performed in serum Opti-MEM medium (Life Technologies), as described (33). DC were washed twice in Opti-MEM and resuspended in Opti-MEM medium at 2–5 × 106 cells/ml and added to 15-ml polypropylene tubes (Falcon). The cationic lipid, N-[1-(2,3-dioleoyloxypropyl]-N,N,N,-trimethylammonium methylsulfate (DOTAP; Boehringer Mannheim, Mannheim, Germany) was used to deliver RNA into cells (33). RNA (25 μg in 250 μl Opti-MEM medium) and DOTAP (50 μg in 250 μl Opti-MEM medium) was mixed in 12 × 75-mm polystyrene tubes at room temperature for 20 min at the RNA:DOTAP ratio of 1:2. The complex was added to DC in a total volume of 2 ml and incubated at 37°C in a water bath with occasional agitation for 2 h. After pulsing, the cells were washed and left for an additional 24 h in culture before FACS analysis, measurement of cytokine in the supernatants, and adoptive transfer. Some RNAs were treated with 400 μg/ml proteinase K (Sigma-Aldrich) or 100 U/ml RNase A (Sigma-Aldrich) or RNase-free DNase I (Life Technologies, Gaithersburg, MD) for 30 min at 37°C. The enzymes were inactivated by incubating samples at 65°C for 15 min.
Adoptive immunization, fungal challenge, and assessment of protection
DC (3–5 × 105/each injection) were injected either s.c. in 20 μl of PBS or i.v. in 0.5 ml of PBS following different protocols, as described in Results. In BM-transplanted recipients, DC were administered s.c. twice, 1 and 7 days after BMT. For infection, a week after the last DC administration, mice were i.v. infected with 106 C. albicans cells of the live vaccine or the virulent strain in 0.5 ml of PBS as described (11). When indicated, reinfection with the virulent strain was done 14 days after the primary infection with the live vaccine strain. Resistance to infection was assessed by quantifying the number of CFU (mean ± SE) per organ and production of Th1 and Th2 cytokines in culture supernatants of Ag-stimulated splenocytes (106 splenocytes stimulated with 105 heat-inactivated C. albicans cells for 48 h), and by enumerating the IFN-γ-producing cells, by ELISPOT assay, as described (11). Total and differential white blood cell counts were done by hemocytometry and by staining blood smears from transplanted mice with May-Grünwald Giemsa reagents (Sigma-Aldrich) before analysis.
For DC analysis, cell surface phenotype was assessed with the following mAb reagents: FITC-conjugated mAb anti-CD11c (N418), anti-CD8α (2.43), anti-I-Adb (34-5-3), anti-CD80 (1G10), and anti-CD86 (GL1) (all from BD PharMingen). Unrelated isotype-matched mAbs were used as control. For assessment of Candida protein expression, DC pulsed with viable yeasts or hyphae or transfected with RNA from yeasts or hyphae were sequentially reacted with the IgM B6.1 mAb, specifically reacting to the phosphomannan component of the fungal cell wall, β-1,2-mannotriose (34, 35) or with polyclonal antiserum from vaccinated mice (36) and the FITC-conjugated goat anti-mouse IgA plus IgG plus IgM (H + L) (Euroclone, Milan, Italy). Before all labeling experiments, FcR blocking was performed by incubating cells with 5% normal mouse serum. For chimerism assessment in BMT, the analysis was done as described (26). For lymphocyte analysis, splenocytes were reacted with FITC-conjugated anti-CD4 (L3T4, clone GK1.5), anti-CD8α (Ly-2, clone 53-6.7), and anti-5E6NK (clone 5E6). Staining with PE-conjugated anti-CD44 (Pgp-1, clone IM7) or anti-CD25 (7D4) mAb (BD PharMingen) was done on gated CD4+ cells. Cells were analyzed with a FACScan flow cytofluorometer (BD Biosciences, Mountain View, CA). Nonviable cells were excluded from analysis by accepted procedures involving propidium iodide and narrow forward-angle light scatter gating. Control staining of cells with irrelevant Ab was used to obtain background fluorescence values. Data were evaluated both as the percentage of positive cells and as median fluorescence intensity (MFI).
Cytokine assays
The levels of IL-2, IL-4, IL-6, IL-10, and IL-12 p70 in culture supernatants were determined by ELISA (R&D Systems, Space Import-Export, Milan, Italy). Cytokine titers were calculated by reference to standard curves constructed with known amounts of recombinant cytokines (from BD PharMingen). The detection limits of the assays were <4 pg/ml for IL-2, <3 pg/ml for IL-4, <15 pg/ml for IL-6, <8 pg/ml for IL-10, and <16 pg/ml for IL-12 p70.
ELISPOT assay
IFN-γ-producing cells were enumerated by ELISPOT assay, as described (37). Briefly, freshly isolated splenocytes or purified CD4+ T cells were cultured (1 × 102–1 × 105 cells/well) in complete medium (RPMI 1640 with 10% FCS, 50 mM 2-ME, and 50 μg/ml gentamicin sulfate) for 18 h in 96-well plates previously coated with rat anti-murine R4-6A-2 mAb. Biotinylated AN-18.17.24 mAb was used as the detecting reagent, avidin-alkaline phosphatase conjugate (Vector Laboratories, Burlingame, CA) was used as the enzyme, and 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt (Life Technologies, Gaithersburg, MD) was used as the substrate. Results were expressed as the mean number of IFN-γ-producing cells (± SE) per 104 cells, calculated using replicates of serial 2-fold dilutions of cells.
RAPD-PCR
Fungal RNA was isolated as described above. SP-DC transfected with yeast or fungal RNA were subjected to RNA extraction by the guanidium thiocyanate-phenol-chloroform procedure, as previously described (11). Before cDNA synthesis, all RNA samples were treated with RNase-free DNase I (Life Technologies). cDNA was synthesized from 1 μg of total RNA according to the manufacturer's directions (Superscript Moloney murine leukemia virus reverse transcriptase; Life Technologies). cDNAs were amplified with random primers derived from the M13 phage core sequence (5′-GAGGGTGGCGGTTCT-3′), known to amplify hypervariable inter-repeat DNA sequences from Candida strains (38). Amplifications were performed in volumes of 25 μl containing 10 ng of cDNA, 10 mM Tris-HCL (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dATP, 2′-dCTP, dTTP, and dGTP, 3 mM magnesium acetate, 10 ng of primer, and 1.5 U of AmpliTaq DNA polymerase. The PCR was performed in a PerkinElmer thermal cycler (model 480; PerkinElmer, Wellesley, MA) with an initial denaturation of 97°C for 3 min, followed by 40 cycles of 20 s at 93°C, 60 s at 50°C, and 20 s at 72°C, and a final cycle of 5 min at 72°C. Amplification products were separated by electrophoresis in 1.5% agarose gels in 1× Tris-acetate-EDTA buffer for 13 h at 2 V/cm. A 100-bp ladder (New England Biolabs, Celbio, Milan, Italy) was run in parallel for approximate PCR product band sizing. Amplification products were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.
Student's t test was used to determine significance of values among experimental groups (significance was defined as p < 0.05). In vivo groups consisted of six to eight animals. The data reported were pooled from three to five experiments.
Maturation and cytokine production by DC upon exposure to Candida yeasts or transfected with yeast or hyphal RNA
To assess whether DC undergo functional maturation upon exposure to viable yeasts or RNA from yeasts or hyphae, the surface expression of MHC class II Ags and of CD80 and CD86 costimulatory molecules was assessed in SP-DC and BM-DC exposed to either type of stimuli. The results showed that the expression of class II Ags and of costimulatory molecules was greatly enhanced in both types of DC upon exposure to yeasts or yeast RNA (Fig. 1⇓). Treatment of yeast RNA with DNase or proteinase did not abolish the induction of costimulatory molecules, as did treatment with RNase (Fig. 2⇓), a finding suggesting that sensitization of DC was mediated by RNA.
Murine DC undergo maturation upon exposure to Candida albicans yeasts or to RNA from yeasts or hyphae. SP-DC or BM-DC were generated as described in Materials and Methods from CD2F1 mice and pulsed with viable yeasts (at DC:fungi ratio of 1:1) or 25 μg total RNA plus 250 μl DOTAP for 2 h at 37°C. Pulsing with viable yeasts was stopped with amphotericin B. Cells were assessed 22 h later for MHC class II Ags, CD80, and CD86 costimulatory Ag expression by immunofluorescence analysis with FITC-conjugated mAb to I-A (clone 34-5-3), to CD80 (clone lG10), and to CD86 (clone GL1) molecules. Unrelated isotype-matched mAbs were used as a control (gray lines). Analysis was performed on a FACScan. Numbers indicate the MFI values.
Effect of different treatments on the ability of yeast RNA to induce DC maturation. Splenic DC were transfected yeast RNA for 24 h at 37°C before the assessment of CD80 (A) and CD86 (B) costimulatory Ag expression by immunofluorescence analysis, as detailed in Fig. 1⇑. Before transfection, some RNAs were treated with 100 U/ml RNase A, 100 U/ml DNase I, or 400 μg/ml proteinase K for 30 min at 37°C. The enzymes were inactivated by incubating samples at 65°C for 15 min. MFI of nontransfected DC was 12 for CD80 and 10 for CD86. ∗, p < 0.05, RNase-treated vs untreated (none) RNA.
In contrast, transfecting with hyphal RNA did not result in up-regulated expression of costimulatory molecules (Fig. 1⇑), a finding suggesting that maturation of DC is a specific effect of yeast RNA and not a consequence of a nonspecific effect of the transfection method itself. The inability of hyphal RNA to sensitize DC was not due to a defective entry of RNA into the cells, as both yeast and hyphal RNA could be detected inside the cells by random amplified polymorphic DNA (RAPD) analysis (Fig. 3⇓). An 867-bp band that was similar in size to that amplified in fungal cDNAs (Fig. 3⇓) or in genomic DNAs from Candida strains (38) was detected in DC transfected with either yeast or hyphal RNA. As higher amounts of hyphal RNA still failed to up-regulate costimulatory molecule expression on DC (data not shown), all together, these data suggest that the induction of DC maturation is a property of yeast RNA.
Detection of fungal RNA in transfected DC by RAPD-PCR. cDNAs from fungal RNA or from SP-DC, after 30 min of transfection with yeast or hyphal RNA, were amplified with random primers, as detailed in Materials and Methods. Amplification products were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. Lane 1, H20 contamination control; lane 2, SP-DC transfected with DOTAP only; lane 3, SP-DC transfected with yeast RNA; lane 4, SP-DC transfected with hyphal RNA; lane 5, cDNA from yeast RNA; lane 6, cDNA from hyphal RNA. bp, DNA ladder.
We have already shown that myeloid DC produce opposite sets of Th cytokines upon exposure to yeasts (IL-12 p70 and IL-6) or hyphae (IL-4) (11). In this study we found that both types of DC produce IL-12 p70 and IL-6, but not IL-4 or IL-10, upon exposure to either yeasts or yeast RNA (Table I⇓). Therefore, these data suggest that both live yeasts and yeast RNA are effective stimuli capable of inducing functional maturation and cytokine production by myeloid DC.
Table I.
Production of cytokines by SP- or BM-DC upon exposure to Candida albicans yeasts or RNA from yeasts or hyphae
Expression of fungal Ags on DC upon exposure to Candida cells or Candida RNA
To assess whether effective transduction of fungal RNA occurs in DC after RNA transfection, we measured the surface expression of fungal proteins by reacting cells with a polyclonal antiserum from vaccinated mice, previously shown to contain Candida-specific Abs (36). We also assessed the levels of expression of mannoproteins, known to induce protective immune responses against disseminated candidiasis (39), with the use of the B6.1 mAb directed to the β-1,2 mannotriose of the phosphomannan protein complex (34, 35). The results show that DC stained positive with either the polyclonal or the mAbs after pulsing with viable yeasts. DC transfected with yeast RNA stained positive with polyclonal Abs and less with the mAb (Fig. 4⇓). In contrast, DC pulsed with hyphae or transfected with hyphal RNA did not show positive staining with either type of Abs. Therefore, these data suggest that the transduction of yeast RNA in DC is responsible for the surface expression of fungal Ags, which may include those endowed with protective efficacy in vivo.
DC express fungal Ags upon exposure to Candida albicans yeasts or yeast RNA. Splenic DC from CD2F1 mice were pulsed with viable yeasts or hyphae (at DC:fungi ratio of 1:1 or 1:10, respectively) or 25 μg total RNA plus 250 μl DOTAP for 2 h at 37°C. Pulsing with viable fungi was stopped with amphotericin B. Cells were assessed 22 h later for fungal Ag expression by immunofluorescence analysis with the IgM B6.1 mAb (thick line) or the polyclonal antiserum from vaccinated mice (thin line) and subsequent reaction with the FITC-conjugated goat anti-mouse IgA plus IgG plus IgM (H + L) (Euroclone). Before all labeling experiments, FcR blocking was performed by incubating cells with 5% normal mouse serum. Unrelated isotype-matched mAbs were used as a control (filled histogram). Analysis was performed on a FACScan.
Immunization with yeast RNA-transfected DC induces Th1-mediated antifungal resistance
DC pulsed with Candida yeasts, but not hyphae, were capable of inducing Th1 priming and antifungal resistance when adoptively transferred into naive recipients (11). To assess whether DC transfected with yeast RNA also induce antifungal resistance in vivo, SP-DC or BM-DC were exposed to yeasts or to RNA from yeasts or hyphae in vitro and injected in vivo into naive recipients following different experimental protocols, which include different routes and timing of DC administration. Mice were subsequently infected i.v. with virulent C. albicans and fungal growth in the kidneys was recorded a week after the infection. The results show that the fungal growth was significantly decreased in mice who had received either type of yeast-pulsed DC administered s.c. 14 days before challenge (Fig. 5⇓). However, consistent with previous data (11), the fungal growth was even more restrained when DC were administered twice, 14 and 7 days before the infection. In contrast, no effect was observed when DC were given 14 days before by the i.v. route. Results with yeast or hyphal RNA-transfected DC showed that the adoptive transfer of yeast, but not hyphal, RNA-transfected DC s.c., twice, also resulted in a significant inhibition of fungal growth (Fig. 5⇓). To understand whether, similar to what had been observed with yeast-pulsed DC (11), the antifungal resistance correlates with the priming of Th1 cells, the production of Th1 (IFN-γ) and Th2 (IL-4 and IL-10) cytokines was evaluated in the spleens of mice who had received multiple s.c. injections of RNA-transfected SP- or BM-DC. The results (Fig. 6⇓) show that production of IFN-γ was higher and that of IL-4/IL-10 was lower in mice who had received multiple s.c. injections of yeast RNA-transfected DC as compared with either mice who had received DC transfected with hyphal RNA or control mice not receiving DC. Together, these data suggest that yeast RNA-transfected DC, similar to yeast-pulsed DC, are capable of inducing Th1 priming and antifungal resistance in vivo, and that the priming capacity is strictly dependent on mode of DC delivery in vivo.
Adoptively transferred DC pulsed with Candida yeasts or transfected with yeast but not hyphal RNA induce resistance to Candida albicans infection. SP-DC or BM-DC were isolated from CD2F1 mice and pulsed as in Fig. 1⇑. A total of 5 × 105 DC were administered to recipient CD2F1 mice, either s.c. 14 (▦) or 14 and 7 (▪) days before infection, or i.v. (▤) 14 days before i.v. challenge with 5 × 105 C. albicans virulent cells. Seven days after infection, quantification of fungal growth was done in the kidneys and expressed as CFU. □, Mice not receiving DC; checked box, mice receiving DC transfected with hyphal RNA; ∗, p < 0.05, mice receiving DC vs mice not receiving DC.
Cytokine production in mice with Candida albicans infection upon adoptive transfer of fungal RNA-transfected DC. SP-DC or BM-DC were isolated and pulsed as in Fig. 1⇑ and injected s.c. twice into CD2F1 mice infected as in Fig. 3⇑. Three days after infection, levels of cytokines in culture supernatants of stimulated with inactivated Candida cells were determined by cytokine-specific ELISA. ▪, DC transfected with yeast RNA; checked box, DC transfected with hyphal RNA; □, mice not receiving DC; ∗, p < 0.05, mice receiving DC vs mice not receiving DC.
Yeast RNA-transfected DC induce antifungal resistance in allogeneic T-depleted BM-transplanted mice
In a mouse model of allogeneic T-depleted BMT, we have recently shown that susceptibility or resistance to candidiasis correlates with the temporal occurrence of Th2 and Th1 cell responses, with Th2 reactivity accounting for susceptibility to the infection in the early engraftment period (26). To assess whether the infusion of Candida-pulsed DC would accelerate Th1 cell recovery and antifungal resistance in transplanted mice, recipients mice received at 1 and 7 days post-BMT donor DC in vitro pulsed with yeasts or yeast RNA. One week after the last DC administration, mice were i.v. infected with the Candida live vaccine strain and 14 days later they were infected with the virulent strain. Mice were monitored for resistance to infection with virulent cells and for parameter of Th1-mediated antifungal resistance, such as the frequency of IFN-γ-producing CD4+ T splenocytes. The results (Fig. 7⇓) showed that the infusion of yeast-pulsed or RNA-transfected DC greatly accelerated the recovery of antifungal resistance, as compared with control mice not receiving DC. A reduced fungal burden was observed in the kidneys and was comparable to that seen in donor-vaccinated mice upon reinfection. Interestingly, the fungal growth was also significantly reduced in organs that are sites of fungal disease and pathology in BMT, such as the gastrointestinal tract, the liver, and the lungs (26). The infusion of yeast-pulsed or RNA-transfected DC also increased resistance toward the primary infection with virulent Candida cells (data not shown). Total and differential counts of blood leukocytes indicated that the absolute number of circulating lymphocytes significantly increased in DC-infused and transplanted mice and was similar to that of donor-vaccinated mice, as shown in Fig. 8⇓ for mice infused with RNA-transfected DC. The number of monocytes and neutrophils did not show significant variations among groups (data not shown). Cytofluorometric analysis of spleen cells revealed that the numbers of CD4+, CD8+, and NK cells were significantly higher in mice who had received RNA-transfected DC as compared with mice not receiving DC and were similar to those of donor-vaccinated mice. Interestingly, CD4+ T cells also stained positive for the CD44 and CD25 activation surface markers (Fig. 8⇓).
Adoptive transfer of Candida-pulsed DC induces resistance to C. albicans infection in a murine model of allogeneic T-depleted BMT. Lethally irradiated C3H/HeJ mice were transplanted with ≥ 2 × 106 T cell-depleted allogeneic BM cells from CD2F1 mice, infected with 106 cells of the live vaccine strain of C. albicans 14 days later, and reinfected with 106 virulent C. albicans cells 2 wk after the primary infection. One and 7 days post-BMT, mice received 5 × 105 DC pulsed with either Candida yeasts (▦) or yeast RNA (▪). ▤, Donor CD2F1 mice vaccinated with 106 cells of the live vaccine strain of C. albicans and reinfected 14 days later with virulent C. albicans strain; □, BM-transplanted mice not receiving DC. At 3 days after reinfection, the fungal growth in different organs was evaluated and expressed as CFU per organ. ∗, p < 0.05, mice receiving DC or donor mice vs mice not receiving DC.
Quantitative and qualitative analysis of lymphocytes in allogeneic T-depleted BM-transplanted mice upon infusion of RNA-transfected DC. Recipient C3H/HeJ mice were BM-transplanted, infused with RNA-transfected DC (▪), and infected as in Fig. 4⇑. ▤, Donor CD2F1 mice vaccinated with 106 cells of the live vaccine strain of C. albicans and reinfected 14 days later with the virulent C. albicans strain; □, BM-transplanted mice not receiving DC. At 3 days after reinfection, peripheral blood lymphocytes were counted by May-Grünwald, and Giemsa staining of blood smears and phenotypic analysis was done by reacting splenocytes with FITC-conjugated anti-CD4, anti-CD8α, and anti-5E6NK mAbs, or, for double staining, with FITC-conjugated anti-CD4 mAb followed by PE-conjugated anti-CD44 or anti-CD25. Cells were analyzed with a FACScan flow cytofluorometer (BD Biosciences). ∗, p < 0.05, mice receiving DC or donor mice vs mice not receiving DC.
On assaying the pattern of cytokine production, we found that the frequency of IFN-γ-producing cells was increased in both freshly isolated T splenocytes and purified CD4+ T cells from mice receiving either yeast-pulsed or RNA-transfected DC as compared with recipients not receiving DC and was similar to that of donor-vaccinated mice (Fig. 9⇓a). Upon Ag stimulation in vitro, splenocytes from DC-infused mice produced levels of IL-4 that were lower than those of mice not receiving DC and were similar to those of vaccinated donor mice (Fig. 9⇓b). Interestingly, however, levels of IL-2 were also lower and those of IL-10 higher in DC-infused mice, as compared with not only untreated recipients but also donor-vaccinated mice. Therefore, the activation of CD4+ lymphocytes producing IFN-γ occurs together with IL-10 but not IL-2 production in hematopoietic BM recipients upon adoptive transfer of Candida-pulsed DC. Experiments in which RNA-transfected DC were infused into recipient mice who had received T-depleted BM cells from IFN-γ−/− mice demonstrated that the efficacy of RNA-transfected DC was mediated by the occurrence of donor IFN-γ-producing cells. Fig. 10⇓ shows that the fungal growth in these mice was comparable to that observed in mice not receiving DC and significantly higher than that of mice transplanted with T-depleted BM cells from IFN-γ-sufficient mice. As a result, similar to what we observed in mice not receiving DC, all the mice succumbed to the infection as opposed to the long-term survival, with no signs of diseases, of mice who had received T-depleted BM cells from IFN-γ+/+ mice.
Adoptive transfer of fungal RNA-transfected DC increases Th1 cytokine production and lowers that of IL-4 in BM-transplanted mice. BM-transplanted mice were generated, infected, and infused with either yeast-pulsed (▦) or RNA-transfected DC (▪) as in Fig. 4⇑. At 3 days after reinfection, the frequency of IFN-γ-producing cells in freshly isolated T or CD4+ splenocytes was assessed by ELISPOT assay (a), and the production of cytokines was measured in culture supernatants of splenocytes upon stimulation with inactivated Candida cells for 48 h (b). □, Mice not receiving DC; ▤, donor CD2F1 mice vaccinated with 106 cells of the live vaccine strain of C. albicans and reinfected 14 days later with the virulent C. albicans strain. Cytokines (measured in picograms per milliliter) were determined by cytokine-specific ELISA. Levels of cytokines in unstimulated splenocytes were below the detection limit of the assays. ∗, p < 0.05, mice receiving DC or donor mice vs mice not receiving DC.
FIGURE 10.
Fungal RNA-transfected DC fail to induce antifungal resistance in mice receiving allogeneic T-depleted BM cells from IFN-γ−/− mice. Lethally irradiated C3H/HeJ mice, transplanted with ≥ 2 × 106 T cell-depleted BM cells from wild-type (▪) or IFN-γ−/− (▧) mice, received 5 × 105 RNA-transfected DC 1 and 7 days post BMT. A week after the last DC administration, mice were infected with 106 C. albicans live vaccine strain and reinfected with virulent Candida cells 14 days later. □, BM-transplanted mice not receiving DC. At 3 days after reinfection, the fungal growth in different organs was evaluated and expressed as CFU per organ. ∗, p < 0.05, mice receiving DC vs mice not receiving DC.
The present study shows that, consistent with previous results obtained with myeloid DC pulsed with Candida yeasts (11), DC transfected with yeast RNA are fully competent to induce Th1 priming upon adoptive transfer in vivo. Induction of antitumor immunity with tumor RNA-transfected DC represents a recently described approach for DC-based vaccines (25). In this study we show that this approach could be used to induce antimicrobial immunity as well.
It has been shown that DC transfected with MUC1 RNA expressed MUC1 tumor Ag along with costimulatory and adhesion molecules (24), a finding suggesting that newly synthesized molecules are potential targets for CTL recognition of RNA-transfected cells (24). We found in this study that DC from both BM and spleens underwent functional maturation in vitro upon pulsing with either live yeasts or yeast RNA, but not with hyphal RNA, as revealed by the up-regulated expression of MHC class II Ags and costimulatory molecules. No variation in CD8α expression was observed upon pulsing with either type of stimulus (data not shown), a finding suggesting that, in our system, myeloid DC do not convert into lymphoid DC upon activation, as suggested (40). Moreover, similar to yeast-pulsed DC, DC transfected with yeast but not hyphal RNA produced IL-12 p70 along with IL-6. As the ability of yeast-pulsed DC to induce Th1 priming in vivo was lost in conditions of IL-12 deficiency (11), these data suggest that DC transfected with yeast and not hyphal RNA would be fully competent to induce Th1 activation.
For this to occur, a prerequisite would be the expression of fungal Ags capable of inducing protective immunity on the surface of DC upon transfection with yeast RNA. This appeared to be the case as DC transfected with yeast but not hyphal RNA positively reacted with polyclonal Abs specifically reacting with Candida cells (36) and, partially, with mAb binding to a specific component of the acid-labile portion of the phosphomannan complex, which is known to be endowed with the ability to induce protective immunity in experimental candidiasis (39).
Pathways and mechanisms of fungal RNA and Ags processing by DC are largely unknown at the moment and remain to be explored.
Upon adoptive transfer in vivo, yeast RNA-transfected DC were capable of inducing the activation of Candida-specific Th1 cells, as revealed by the predominant production of IFN-γ over that of IL-4/IL-10. Moreover, the IL-12β2R, a marker of CD4+ Th1 cells in candidiasis (5), was expressed on CD4+ T lymphocytes (data not shown). Associated with the occurrence of Candida-specific Th1 reactivity was the induction of a state of antifungal resistance, as revealed by the significant decrease of fungal growth in the target organs, such as the kidneys and the brain (data not shown).
Previous work in vivo in mice and in humans has suggested that, in addition to the maturation state (41), the mode of delivery of DC is important in eliciting T cell responses (42, 43, 44, 45). DC given i.v. localized in the reticuloendothelial system and lung, while those given s.c. preferentially home to the T cell areas of the draining lymph nodes (42). In particular, induction of IFN-γ production, but not IL-4, was seen only with DC injected intradermally or intralymphatically but not i.v. (43). We found a similar result, as Th1 cell activation was observed after s.c. but not i.v. administration of DC. Although multiple injections of DC could lead to clonal exhaustion of the responding T cell population (43), the optimal protection against C. albicans was observed after two s.c. injections of Candida-pulsed DC. Labeling studies will clarify whether DC loaded with Candida home to different organs upon administration by different routes and whether the extent to which the ability of DC to migrate to relevant lymphoid organs will regulate the type and magnitude of the anticandidal immune response.
An interesting observation of the present study was that the infusion of Candida-pulsed DC accelerated the recovery of functional antifungal Th1 responses in a murine model of T cell-depleted allogeneic BMT. Patients receiving T cell-depleted BMT are unable to develop Ag-specific T cell responses soon after transplant (46). It has been demonstrated that T cell depletion of allogeneic BMT is associated with a slow recovery of CD4+ and CD8+ T cells (46). However, functional recovery of the T cell system after T cell-depleted allogeneic BMT has been demonstrated (47, 48), and both donor and recipient DC may contribute to the reconstitution of the T cell repertoire in transplantation through distinct pathways of Ag presentation (49). Interestingly, it has been reported that T cell reactivity to Candida readily recovered after T cell-depleted allogeneic BMT, and the fact that recovery was related to the absence of antifungal prophylactic measures indicates that anticandidal reactivity of residual T cells could be maintained through continuous exposure to the fungus (50).
We have recently demonstrated that an unbalanced production of Th1 and Th2 cytokines was responsible for the susceptibility to candidiasis observed in our BMT model. However, readdressing the balance between Th1 and Th2 subsets, as by treatment with Th2 cytokine antagonists, accelerated the recovery of Th1-mediated antifungal resistance (26). In this study we show that recovery of functional NK and T cells producing IFN-γ could also be accelerated by the infusion of Candida-pulsed DC. This, along with the decreased IL-4 production, translated in the occurrence of a state of antifungal resistance as revealed by the impaired fungal growth in different target organs, including those most frequently associated with fungal disease and pathology in BMT settings, such as the gut, liver, and lungs. Therefore, although conflicting data exist as to whether donor DC are "friends" or "foes" in transplantation (49), our findings suggest that DC may contribute to the educational program of T cells in BMT during reconstitution, in line with the recent observation of the immunization capacity of tumor-pulsed (51) or Aspergillus-pulsed (18) DC in BM-transplanted mice and humans.
The observation that the occurrence of a protective Th1 reactivity coexisted with the detection of significant levels of IL-10 is intriguing. It is known that high levels of IL-10 are associated with tolerance to HLA-mismatched BM stem cells (52) and IL-10 is required for the induction of regulatory T cells mediating tolerance to alloantigens in vivo (52, 53, 54, 55). Different regulatory T cells have been reported and they are known to differentiate upon the encounter with immature DC (53, 55). However, fully competent DC were also found to be inducers of T cell anergy when presenting a self-epitope with altered peptide ligand features (56). Whether IL-10 produced in BM-transplanted mice infused with Candida-pulsed DC may serve to support the growth of regulatory T cells preventing donor Th1 alloreactivity remains a working hypothesis, although it may help to explain the long-term, disease-free survival of the mice. In terms of interference with the development and activity of antifungal Th1 reactivity, it is well established that the effect of IL-10 is dependent upon the dose of the cytokine. At high doses, IL-10 had a negative effect on Th1-mediated antifungal resistance (57), but at low doses IL-10 was required for optimal induction and maintenance of anticandidal Th1 immune response (58).
A recent study showed that yeast cells could act as an efficient vaccine and elicited protective cell-mediated immunity by virtue of their ability to induce DC maturation, IL-12 production, and efficient priming for MHC class I- or II-restricted Ag-specific T cell responses (59). We show in this work that not only the yeasts but yeast RNA could be an efficient trigger of DC maturation, IL-12 production, and induction of protective Ag-specific responses to Candida. Interestingly, inactivated yeasts failed to induce DC maturation in vitro, and DC pulsed with inactivated yeasts failed to promote Th1 immunity upon adoptive transfer in vivo (data not shown). Therefore, these data may account for the long-standing observation of the inability of inactivated Candida to induce memory anticandidal protective immune responses.
It has been shown that human DC phagocytose and process C. albicans (60, 61) as well as other fungi (18, 62), and ex vivo-generated DC pulsed with Aspergillus conidia restored antifungal immunity in BM-transplanted patients (18). This work suggests that DC could act as effective vaccines against fungal infections and that RNA-transfected DC could be of vaccinating potential in conditions that negate the use of attenuated microorganisms, such as immunosuppression, or in the case of poor availability of protective Ags.
We thank Dr. Lara Bellocchio for dedicated editorial assistance, Andrea Casagrande and Sabrina Fiorucci for their dedicated work in the laboratory, and Carla Barabani of the animal facility at the University of Perugia for technical assistance.
↵1 This work was supported by the National Research Project on AIDS (contract 50C.27, "Opportunistic Infections and Tuberculosis").
↵2 Address correspondence and reprint requests to Dr. Luigina Romani, Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. E-mail address: lromani{at}unipg.it
3 Abbreviations used in this paper: BMT, bone marrow transplantation; DC, dendritic cell; BM, bone marrow; BM-DC, BM-derived DC; SP-DC, spleen-derived DC; RAPD, random amplified polymorphic DNA; DOTAP, N-[1-(2,3-dioleoyloxypropyl]-N,N,N,-trimethylammonium methylsulfate; MFI, median fluorescence intensity.
Received September 10, 2001.
Accepted January 9, 2002.
Copyright © 2002 by The American Association of Immunologists
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You are going to email the following Dendritic Cells Pulsed with Fungal RNA Induce Protective Immunity to Candida albicans in Hematopoietic Transplantation
Angela Bacci, Claudia Montagnoli, Katia Perruccio, Silvia Bozza, Roberta Gaziano, Lucia Pitzurra, Andrea Velardi, Cristiana Fe' d'Ostiani, Jim E. Cutler, Luigina Romani
The Journal of Immunology March 15, 2002, 168 (6) 2904-2913; DOI: 10.4049/jimmunol.168.6.2904
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Boy Suffers Near Fatal Spine Injury Attempting to Imitate Uzumaki
January 24, 2016 - News - Tagged: Junji Ito, Local, Manga, Uzumaki
The playground where the incident allegedly took place
BOYS TOWN, NE– Last week Kyle Hansford, 12, twisted his entire body into a spiral at his middle school playground. The child was rushed to the local hospital and after an experimental operation known as the 'Fruit Unroll Up', the boy was moved out of critical condition.
Kyle's injury was an imitation of a scene from the Junji Ito's manga Uzumaki, in which a man stuffs his body inside a pot, twisting himself into a spiral-like shape. The manga has been praised by critics and fans alike, but everyone agrees that scene was pretty gross.
I tried once, but I could never reach.
According to playground hearsay, Kyle had been dared into the act. While Kyle refused initially, he was then reportedly double-dog-dared to imitate the famous scene. Now with a double-dog-dare on his hands, Kyle had no choice but to attempt the feat. Unfortunately after getting his head a quarter way around, Kyle's body locked up. The boy was stuck in a spiral, and immediately abandoned by his friends, who feared repercussions from their teacher.
Kyle's parents were distraught at the revelation of their son's pretzel predicament. Upon discovering the inspiration for the incident Debra Hansford, Kyle's mother, called the award-winning manga 'pornographic', 'disgusting', and 'very rude'. She has even taken measures to attempt banning Uzumaki from stores, even organizing book burnings. Mrs. Hansford has gained a sizable following on Common Sense Media.
"Why should I take responsibility for my child's actions when it's clearly the fault of these Chinese comic books?" Mrs. Hansford exclaimed in front of a gathered crowd while holding up a copy of Naruto.
As of press time, no other spiral related incidents have been reported. Although authorities in Boys Town are on the lookout for human sized snails.
Talking about himself in the third person has never be Morlock's strengths, however it would seem that duty calls for a change in POV. Hobbies include: self loathing, mindlessly consuming japanese media like the weeaboo that he is, and complaining. Mad Bull 34 is still the greatest anime Morlock has seen. For some reason he has a Twitter: @TheMorlock


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Donate / Events
Our K9 Teams
These are just some of our amazing K9s
Team Shylo Handler: Jimi
K9 Shylo is a 4 Year old Black and Tan, German Shepard and is one of our operational K9s. She has been with South Central PA Search and Rescue for 4 years and was in training as soon as she joined the team. She weighs 65 pounds and enjoys her play time when she is not training. She is trained to do Scent work, trail and tracking. She has attended several Public Relation events and Educational events during the past four years. K9 Shilo completed, and received her certificate of training from the INTBI, (Inter-National Bloodhound Training Institute) Man trailing Seminar, 27 – 29 Sep 2019. K9 Shylo is also CGC Certified. K9 Shylo has been to several of the teams call outs over the last four years and has taken over for K9 Sadie whom has retired from active search call outs. K9 Shylo continues to train and works at several PR Events as well.
Team Charlotte Handler: Jen
K-9 Charlotte is a dual-certified search and rescue dog She is certified in trailing and human remains detection Charlotte has completed her canine good citizen and is also a member of the precious paws therapy dog program. Charlotte is completed her certifications through Pennsylvania search and rescue council and the national association of Search and Rescue otherwise known as nasar. Charlotte has attended many seminars throughout the United States and is also enlisted in a program through CUE which is the center for missing and exploited children based out of North Carolina. Charlotte has also been listed as one of the top three search and rescue dogs in the United States through a nomination through American humane hero dog awards in 2019.
Team Jules Handler: Chris
K9 Jules is a 4 yr old German Shepherd mix and is one of our operational trailing K9s. She has been with South Central PA Search and Rescue since November 2019. When she is not training she enjoy supervising her neighborhood and handler while he is working in his garden. She has trained with instructors from INBTI(International Bloodhound Training Institute) and NAPWDA(North American Police Work Dog Association).
Team Puck Handler: Laurie
K9 Puck, is a four year old, black sable, long coat, working line German shepherd. He is trained and works as a non-scent specific, area search air scent K9. When he is not working as a Search and Rescue he is, as his handler Laurie states, "first and foremost a beloved family member, who lives with his handler." His sister, K9 Mali and a few other pet dogs share his home with his humans.
K9 Puck is a medium drive K9 that was intended to do K9 sports, however, his drive was higher and once introduced to Search and Rescue work he found his knitch. This work matches his drive and desire and he is always ready to work when needed.
K9 Puck has his AKC CGC (Canine Good Citizen) and is working toward a therapy dog certification. He is trained in competition obedience and competes in dock diving. Being a K9 partner runs in Puck's family, as Puck's father was his handler's previous partner, Var. He has been a member of the team for three years.
Team Mali Handler: Keith
K9 Mali is an eight year old, dark sable, stock coat working line German Shepherd dog. She is working as a scent specific tracking & trailing dog. She is a very high drive and high energy dog who is on a second career doing search and rescue. Her retirement from sport did not suit her. She has a drive that is only answered by working the trail and training with the K9 team. Mali has actually earned her AKC CGC (Canine Good Citizen) twice, however, her handler lost the original paperwork.
K9 Mali is a titled competition dog and still does competition obedience and agility and soon dock diving.
Above all else Mali is a beloved family member, she who will work for food, a ball or a play tug, praise is always part of the equation. So far Mali has done very well at everything asked of her except sitting still. K9 Mali has been a member on the team for one year and continues to make great progress.
Copyright © 2022 Search93-SCPASAR - All Rights Reserved.


Request a spot for your child in the Preschool Program.
What do you think they will be when they grow up?
They work on holding a pencil properly, tracing letters, distinguishing between upper and lower case letters and recognizing the letters of their names. They even begin to read short words.
Our preschool program has a 8:1 student to teacher ratio, which means your child will get as much attention as he or she needs.
If you want to enrol your child into our PreSchool Program, the first step is booking a tour of the facility for you and your child. If you think it's the right fit, the next step is requesting a spot. If no spot is available, your child will be put on a wait list.
Please note: requesting a spot is not equivalent to submitting an application. Applications are only delivered if spots are available, otherwise your child will be added to a wait list. Click Here View our Admission and Wait List Policy.


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ALBERT STUBBINS – THE SERGEANT PEPPER MAN
by Vince Cooper | Nov 1, 2017 | The Big Feature
THE cover of The Beatles' Sergeant Pepper's Lonely Hearts Club Band album gave lots of fun for those looking to spot which famous personalities had made the 'cut'. The likes of Karl Marx, Bob Dylan, Laurel & Hardy and Marilyn Monroe were included in the 70 featured and there were also some lesser-known faces. From the sporting world boxer Sonny Liston is easy to identify, swimming star and Tarzan man Johnny Weissmuller perhaps less so. And then there is the footballer.
Sonny, Johnny…..and Albert
Released in the year after England's World Cup win you might have expected Bobby Moore, Geoff Hurst or maybe even Eusebio or Pele, the biggest names in world football at the time, to figure on the cover of the world's biggest band's latest masterpiece. Instead it was Albert Stubbins who featured as the sole representative of the Beautiful Game, his smiling face peeking out from behind Marlene Dietrich, and he surely would have caught out many trying to name them all.
A Liverpool great from the post-war era, Stubbins was, apparently, a John Lennon choice for the cover although none of the Fab Four can be said to have been massive football fans. In fact Lennon's reason for choosing him for the cover was said to be that the name had amused him as a child.
Born in Wallsend on Tyneside in 1919, Stubbins spent part of his childhood in the United States but his family returned home and he first signed amateur forms for Sunderland although only on the understanding that if Newcastle wanted him he would be allowed to make the switch. The Geordies duly made their move and he made his.
The well-built striker had his initial taste of first team action in 1937 and he was just getting into his stride for Toon when World War Two broke out.
Stubbins was a prolific scorer in War-time football, although sadly the almost 200 goals he got in that period don't count towards official records. Maintaining his 'other' job as a draughtsman he turned out regularly for his club and such was his form that he was chosen for England in the 1945 Victory International against Wales at The Hawthorns .
The Welsh recorded a shock 1-0 victory that day. Stubbins was never picked for his country again and with the match not classified as a full international this meant he never won an official cap.
In the first season after the war, and before the Football League resumed activities, The centre-forward scored 39 times for Newcastle in the Northern League. By this time the club had another promising forward emerging in a certain Jackie Milburn and it might well have been to the benefit of all when Stubbins asked for a transfer.
Spin The Coin
Newcastle made their star man available and no fewer than 18 clubs registered an interest. The top two suitors, Liverpool and Everton, both had bids of around £12,500 accepted. And the story of how Stubbins made his choice between the two deserves retelling.
"Both teams [Liverpool and Everton] wanted to talk to me and it was a very difficult decision to make," recalled Albert. "During the War we had a few guest players from Liverpool at Newcastle and so I suppose I always had a slight inclination towards Liverpool.
"Everton though were also a famous team at the time and were known as "The team of talents"."
Albert was enjoying a night out at the cinema when a notice appeared on the screen. 'Would Albert Stubbins please report to St James' Park'.
When he arrived at the stadium representatives of both clubs were present and Newcastle director Stan Seymour asked who he would like to see first. ''I said let's flip a coin, 'Heads Liverpool. Tails Everton.' It came down heads – Liverpool.
"Bill McConnell, the Liverpool chairman and George Kay [the manager] and myself discussed matters and I was impressed with them both, and with the possibilities of Liverpool, so I agreed to go to Anfield.
"In the end I never spoke to Everton because I had been so impressed with Liverpool's offer".
It Was 70(ish) Years Ago Today
The new man settled in quickly at Anfield. He formed an excellent partnership with Jack Balmer, the pair finishing joint-top scorers in the league in the 1946-47 season with 24 goals apiece.
With further support from Billy Liddell, Bob Paisley and others the Reds sneaked to the first post-war title that season coming out on top in a tight, thrilling race where they, Manchester United, Wolverhampton Wanderers and Stoke City were separated by a mere two points.
After another fine season where he scored 26 goals, things suddenly seemed to go wrong. At one stage he asked Liverpool for a transfer but was refused. He helped them to the 1950 FA Cup Final but played with an injury and his team lost to Arsenal and in a further five seasons at Anfield, managed just 27 goals after scoring 50 in his first two.
He did get a second chance at representative football in 1950 when chosen to play for the Football League against the Irish League at Bloomfield Road and certainly didn't let the side down, scoring five times in a 6-3 win.
Thereafter injuries took their toll. He left Liverpool in 1953 and spent a season with Ashington after being encouraged to join the club by Jackie Milburn.
After retirement Stubbins became a highly-respected sports journalist although he did return to the game briefly to coach the New York Americans, a team of expats playing in the U.S International Soccer League. He died aged 82 in 2002 after a short illness.
Albert Stubbins received a copy of Sergeant Pepper's Lonely Hearts Club Band and a thank-you note from Paul McCartney when the album was released. He also received a League winners medal and an FA Cup runners-up medal from his playing days. And a guaranteed place in immortality thanks to that unlikely source.
« FOOTBALL OR CRICKET? BOTH GONE GROUNDS – LEEDS ROAD »
GONE GROUNDS - LEEDS ROAD
FOOTBALL OR CRICKET? BOTH


Have a strong background in cloud security engineering? Looking for a change in jobs going into the new year. My client based out of the Atlanta area is looking to bring on a Cloud Security Engineer for an upcoming project set to begin around the New Year.


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28February201828th August 2021
Artist SpotlightBy Artvisor
Artist Spotlight: Getulio Alviani
Born: Udine, Italy, 1939.
After a long battle with illness, the acclaimed Italian artist, author and designer Getulio Alviani died on the 24th February 2018, in his home city of Milan, aged 78. Alviani, who was considered a protagonist of Optical and Kinetic Art, leaves behind a rich and prestigious legacy.
Having become interested in art (and particularly geometric drawing) at a young age, Alviani gradually channelled his creative focus towards design-oriented approaches to painting and sculpture. His curiosity for new technologies and innovation was prominent throughout his artistic career.
Getulio Alviani, Superficie testura vibrante (Vibrant Textured Surface) (1969). Image courtesy artist and gallery.
Alviani and post war technological innovation
During the 1950s, Alviani closely followed the pursuits of contemporary architects and engineers and he transferred much of the influences he took from them into his sculptural practice. His aluminium pieces, such as the famous The Wires series, which drew inspiration from aerial electrical wires, received immediate recognition for their dynamic minimalist forms. Though many of his peers revered the era's technological advancements, Alviani focused not on their novelty but on the connection he could form between technology and his artistic practice.
By the 1960s, with his participation in the 'Arte Programmata,' or Programmed Art, shows in Venice, Rome, Dusseldorf and Leverkusen, Alviani's international reputation was accelerating. He went on to exhibit at the 1964 Venice Biennale, and in 1965 was featured in the renowned exhibition, 'The Responsive Eye,' at Moma, New York.
Installation shot, 'Getulio Alviani,' (2006) Gallery Vistamare Mala di Lubiana, Pescara, Italy. Image courtesy artist and gallery.
Alviani's metallic sculptures shimmer with chromatic intensity; their patterned structures reflect an abstracted labyrinth of colour. In doing so, they mirror the act of seeing. His 'Superficie a testura vibratile,' or Vibrating Texture Surface series (circa 1970), for example, exaggerate the transience of light, movement and reflection upon geometric surfaces. These artworks engage and disorientate the viewer, calling into question notions of perception.
Nouvelle Tendance Recherches Continuelles
Alviani had a pedagogical vision for art in society. He believed exhibition spaces should be used for renewing our understanding of cultural and scientific phenomena as well as for sensory experiences. Alviani was a professor of painting at the Academy of Fine Arts of Carrara, a member of the international movement "Nouvelle Tendance Recherches Continuelles" (New Tendencies). Additionally, he was the director of the Museum of Modern Art in Ciudad Bolivar, Venezuela, where he dedicated himself to editing texts and curating exhibitions on the leading figures of visual research at an international level.
Getulio Alviani presented works in important exhibitions, including the founding of Arte Programmed in the Olivetti store in Milan,1962; the 1964 Venice Biennale; the historic 'The Responsive Eye' at the MoMa, New York, in 1965; and more recently, in 2013, 'Dynamo' at the Grand Palais in Paris, which brought together all the protagonists of Kinetic art. MoMa's exhibition, 'Thinking Machines: Art and Design in the Computer Age, 1959-1989,' 2017, which showed Alviani's work in relation to the advent of new information technologies, was received with particular admiration.
Author: George Greenhill
Cover image: Getulio Alviani, Testura Grafica X Ortogonale (1972). Image courtesy artist and gallery.
Artist Spotlight: Justin Fitzpatrick'Germany is not an Island': a timely, hybrid display of contemporary art in 2018


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Press Coverage and Troupe News
Current Charity
Charity Selection Process
Past Charities
The Blue Hill Troupe Announces its 92nd Season to Benefit Children's Cancer & Blood Foundation
Nonprofit Troupe Takes Top Honors at International Gilbert & Sullivan Festival
NEW YORK, NY, September 29, 2015 – The Blue Hill Troupe, the only musical theater group in New York City to donate its net proceeds to charity, announces its 92nd season with A Little Night Music, November 13-21, 2015 at The Theatre at St. Jean's (184 E. 76 Street) and Gilbert & Sullivan's Iolanthe, or the Peer and the Peri, April 8-16, 2016 at El Teatro of El Museo del Barrio (1230 Fifth Avenue). The Blue Hill Troupe's 2015-2016 season will benefit the Children's Cancer & Blood Foundation.
Tickets to A Little Night Music are on sale now and range from $29.50 – $75.00. (Payment in excess of $20 per ticket is tax deductible to the extent provided by law.) They can be purchased at https://bht.org/events/2015-a-little-night-music or by calling 212-988-2012. Performances are November 13, 14, 18, 19, 20 and 21 at 7:30 PM, November 15 at 3:00 PM, and November 21 at 2:00 PM. "A Little Night Music" is directed by Donald Brenner, with music direction by Eric Peterson. The cast features Patricia Berg*, Denise Crawfort, Christopher Dorre, Julia Farina, Sarah Glaser, Sarah Gilbert, Ken Harmon*, Joanne Lessner*, David Loewy, Michael Macaione*, David Pasteelnick, Chazmond J. Peacock, Sydney Ransom, Vikki Willoughby and Cristiane Young. (*Appearing courtesy of Actors' Equity Association.)
The Blue Hill Troupe recently returned from the 23rd Annual International Gilbert & Sullivan Festival in Harrogate, England, where 50 members of the Troupe recreated last season's production of Patience. The Troupe was honored to earn the Festival's 2015 International Champions Award, as well as awards for Best Director, Best Supporting Actor, Best Female Voice, and Best Animated Chorus.
Every year since 1924, the Blue Hill Troupe has come together for a season of revelry celebrating its three great passions: Theater, Charity and Community. The Troupe's efforts are rooted in a tradition of performing a Gilbert & Sullivan operetta, and the group also presents a musical theater production as well as a concert series. The all-volunteer Blue Hill Troupe spans all generations and all walks of life. New members, both front and backstage, are accepted each fall at the beginning of the season and are immediately folded into the family where everyone, from singers to sewers alike, works side-by-side.
Over the years, these efforts have raised more than four million dollars in net proceeds that have been given annually to various diverse NYC-based charities. Recent charities include Quality Services for the Autism Community, the GO Project, Carter Burden Center for the Aging, and the "I Have a Dream" Foundation – New York.
Set in 1900s Sweden, Stephen Sondheim and Hugh Wheeler's elegant gem A Little Night Music explores the tangled web of affairs centered around actress Desirée Armfeldt, the men who love her, and their jealous wives. When all convene for a weekend in the country at Desirée's mother's estate, infinite possibilities for new romances and second chances bring endless surprises.
In Gilbert & Sullivan's Iolanthe, the title character, a fairy, returns to the fold 25 years after being banished from Fairyland for marrying a mortal. She seeks protection for her now grown son who is half-fairy and half-mortal. When he falls in love with the ward of the Lord Chancellor, both Fairyland and the Houses of Parliament nearly fall to pieces.
The Children's Cancer and Blood Foundation (CCBF) was created in 1952 to support the extraordinary work of the Division of Pediatric Hematology and Oncology. It is the oldest and largest charitable organization in the United States dedicated to funding clinical care, research and training of emerging practitioners and researchers in pediatric hematology and oncology. Support from CCBF has fueled many of the Division's breakthroughs in scientific and clinical research. It has revolutionized the treatment of young patients who, as a result, are receiving better care and living longer and fuller lives. Its team prides itself on providing outstanding, innovative, family-centered patient care and recognizes that medical treatment is only one part of the child's road to recovery. CCBF's distinguished researchers are making medical advances that are to revolutionizing the study and treatment of pediatric cancers and blood disorders. CCBF scientists are applying research breakthroughs to practical treatments for patients across the world. (www.childrenscbf.org)
For more information about the Blue Hill Troupe, please visit: www.bht.org, Facebook.com/BHT.org and @BlueHillTroupe.


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On 20th June 2021 By tfncruz In Ofir, Fão, Portugal, Portugal
Ofir, Portugal, 05.2020
For a long time I don't update this website. I will do it more often in order to synchronise it with my instagram! These two drawings were made in my hometown "Ofir" in last may 2020.
Fão
On 10th April 2021 By tfncruz In Uncategorised
waiting Naz
On 1st August 2020 By tfncruz In _other places, Portugal
ESMAD, Vila do Conde, Portugal
On 29th July 2020 By tfncruz In _other places, Portugal
On 26th July 2020 By tfncruz In Ofir, Fão, Portugal, Portugal
Ofir, 27.06.2020
On 14th May 2020 By tfncruz In Ofir, Fão, Portugal, Portugal
Fão, Portugal, 30.05.2020
It is always a difficult decision to make in the iPad… with or without color. On one hand I love the linework. On the other hand, the color has this great capacity to group elements giving the image a more understandable look and feel.
There is a third option. To ignore the line and show the color alone. Like that we go to a more (also interesting) abstract expression.
I have a love/hate relationship with this characteristic of the digital media. This big amount of options to choose from. It sounds good, and it is, but on the other hand, it is much more easy to fall in that deep hole where there is no end for the artwork because of this feeling that all the options are still open for debate and reflexion.
It is more easy to put an end to things when I'm on paper or with other physical media. Less options, less undo, less going back, and this feeling that "I'm missing something that could make this even better". Well, there is always something that could make something better… Something to think about.
Fão, Portugal, 28.4.2020
On 31st March 2020 By tfncruz In Ofir, Fão, Portugal, Portugal
Jamaica and Naz sleeping
rebel princess cooking
Tools can greatly change the way we draw. Here, the slippery surface results in a very interesting wandering line. I love drawing on the iPad…