Mechanistic Insights into Synergy between Melanin-Targeting Radioimmunotherapy and Immunotherapy in Experimental Melanoma.

Melanoma incidence continues to rise, and while therapeutic approaches for early stage cases are effective, metastatic melanoma continues to be associated with high mortality. Immune checkpoint blockade (ICB) has demonstrated clinical success with approved drugs in cohorts of patients with metastatic melanoma and targeted radionuclide therapy strategies showed promise in several clinical trials against various cancers including metastatic melanoma. This led our group to investigate the combination of these two treatments which could be potentially offered to patients with metastatic melanoma not responsive to ICB alone. Previously, we have demonstrated that a combination of humanized anti-melanin antibody conjugated to 213Bismuth and anti-PD-1 ICB reduced tumor growth and increased survival in the Cloudman S91 murine melanoma DBA/2 mouse model. In the current study, we sought to improve the tumoricidal effect by using the long-lived radionuclides 177Lutetium and 225Actinium. Male Cloudman S91-bearing DBA/2 mice were treated intraperitoneally with PBS (Sham), unlabeled antibody to melanin, anti-PD-1 ICB, 177Lutetium or 225Actinium RIT, or a combination of ICB and RIT. Treatment with anti-PD-1 alone or low-dose 177Lutetium RIT alone resulted in modest tumor reduction, while their combination significantly reduced tumor growth and increased survival, suggesting synergy. 225Actinium RIT, alone or in combination with ICB, showed no therapeutic benefit, suggesting that the two radionuclides with different energetic properties work in distinct ways. We did not detect an increase in tumor-infiltrating T cells in the tumor microenvironment, which suggests the involvement of alternative mechanisms that improve the effect of combination therapy beyond that observed in the single therapies.

The adjuvant effect of melanin is superior to incomplete Freund's adjuvant in subunit/peptide vaccines in mice.

Peptide vaccines represent an attractive alternative to conventional anti-tumor therapies, but have not yet achieved significant clinical efficacy with commonly used formulations. Combination of short antigenic peptides, synthetic melanin and TLR9 agonist (Toll-like receptor 9, CpG-28) was reported as highly efficient to trigger strong CD8 + T-cell responses. We compared this vaccine approach to the standard adjuvant formulation that combines the incomplete Freund's adjuvant (IFA) and CpG-28, using either an ovalbumin epitope (pOVA30) or a spontaneously occurring tumor neoepitope (mAdpgk).Melanin-based vaccine induced significantly higher cytotoxic T lymphocytes (CTL) responses than IFA-based vaccine in both pOVA30- and mAdpgk-targeted vaccines. The anti-tumor efficacy of melanin-based vaccine was further assessed in mice, grafted either with E.G7-OVA cells (E.G7 cells transfected with ovalbumin) or with MC38 cells that spontaneously express the mAdpgk neoepitope. Melanin-based vaccine induced a major inhibition of E.G7-OVA tumor growth when compared to IFA-based vaccine (p < 0.001), but tumors eventually relapsed from day 24. In the MC38 tumor model, no significant inhibition of tumor growth was observed. In both cases, tumor escape appeared related to the loss of antigen presentation by tumor cells (loss of ovalbumin expression in E.G7-OVA model; poor presentation of mAdpgk in MC38 model), although the CTL responses displayed an effector memory phenotype, a high cytolytic potential and low programmed cell death-1 (PD1) expression.In conclusion, synthetic melanin can be efficiently used as an adjuvant to enhance T-cells response against subunit vaccine antigens and compared favorably to the classic combination of IFA and TLR9 agonist in mice.

Long-term post-mortem studies following neurturin gene therapy in patients with advanced Parkinson's disease.

We performed post-mortem studies on two patients with advanced Parkinson's disease 8 and10 years following AAV2-neurturin (CERE120) gene therapy, the longest post-mortem trophic factor gene therapy cases reported to date. CERE120 was delivered to the putamen bilaterally in one case (10 years post-surgery), and to the putamen plus the substantia nigra bilaterally in the second (8 years post-surgery). In both patients there was persistent, albeit limited, neurturin expression in the putamen covering ∼3-12% of the putamen. In the putamen, dense staining of tyrosine hydroxylase-positive fibres was observed in areas that contained detectable neurturin expression. In the substantia nigra, neurturin expression was detected in 9.8-18.95% and 22.02-39% of remaining melanin-containing neurons in the patient with putamenal and combined putamenal and nigral gene delivery, respectively. Melanized neurons displayed intense tyrosine hydroxylase and RET proto-oncogene expression in nigral neurons in the patient where CERE120 was directly delivered to the nigra. There was no difference in the degree of Lewy pathology in comparison to untreated control patients with Parkinson's disease, and α-synuclein aggregates were detected in neurons that also stained for neurturin, RET, and tyrosine hydroxylase. These changes were not associated with antiparkinsonian benefits likely due to the limited neurturin expression. This study provides the longest term evidence of persistent transgene expression following gene delivery to the CNS and the first human results when targeting both the terminal fields in the putamen as well as the originating nigral neurons.

Aspergillus fumigatus Cell Wall Promotes Apical Airway Epithelial Recruitment of Human Neutrophils.

Aspergillus fumigatus is a ubiquitous fungal pathogen capable of causing multiple pulmonary diseases, including invasive aspergillosis, chronic necrotizing aspergillosis, fungal colonization, and allergic bronchopulmonary aspergillosis. Intact mucociliary barrier function and early airway neutrophil responses are critical for clearing fungal conidia from the host airways prior to establishing disease. Following inhalation, Aspergillus conidia deposit in the small airways, where they are likely to make their initial host encounter with epithelial cells. Challenges in airway infection models have limited the ability to explore early steps in the interactions between A. fumigatus and the human airway epithelium. Here, we use inverted air-liquid interface cultures to demonstrate that the human airway epithelium responds to apical stimulation by A. fumigatus to promote the transepithelial migration of neutrophils from the basolateral membrane surface to the apical airway surface. Promoting epithelial transmigration with Aspergillus required prolonged exposure with live resting conidia. Swollen conidia did not expedite epithelial transmigration. Using A. fumigatus strains containing deletions of genes for cell wall components, we identified that deletion of the hydrophobic rodlet layer or dihydroxynaphthalene-melanin in the conidial cell wall amplified the epithelial transmigration of neutrophils, using primary human airway epithelium. Ultimately, we show that an as-yet-unidentified nonsecreted cell wall protein is required to promote the early epithelial transmigration of human neutrophils into the airspace in response to A. fumigatus Together, these data provide critical insight into the initial epithelial host response to Aspergillus.

Solubilized melanin suppresses macrophage function.

Melanin-producing Cryptococcus and Aspergillus are highly invasive and can suppress or escape the immune system of the host. Since non-melanin-producing strains do not affect the immune system, melanin may play a role in immune system suppression. Artificial melanin synthesized using conventional methods is insoluble, making structural and functional analysis of this chemical difficult. In this study, we describe a melanin solubilization method based on polymerization of homogentisic acid (solubilizing component) and an equivalent amount of L-DOPA in the presence of laccase. In addition, we investigated the effect of melanin on the immune system. Homogentisic acid and L-DOPA mixed melanin (HALD), the synthetic solubilized melanin, did not exert a cytotoxic effect on mouse macrophages. HALD suppressed cytokine and reactive oxygen species production by macrophages when they were stimulated by fungal components. HALD also suppressed the phagocytosis of fungal components by macrophages. These results suggest that HALD can suppress the function of macrophages without causing cytotoxicity.

Manduca sexta hemolymph protease-2 (HP2) activated by HP14 generates prophenoloxidase-activating protease-2 (PAP2) in wandering larvae and pupae.

Melanization is a universal defense mechanism of insects against microbial infection. During this response, phenoloxidase (PO) is activated from its precursor by prophenoloxidase activating protease (PAP), the terminal enzyme of a serine protease (SP) cascade. In the tobacco hornworm Manduca sexta, hemolymph protease-14 (HP14) is autoactivated from proHP14 to initiate the protease cascade after host proteins recognize invading pathogens. HP14, HP21, proHP1*, HP6, HP8, PAP1-3, and non-catalytic serine protease homologs (SPH1 and SPH2) constitute a portion of the extracellular SP-SPH system to mediate melanization and other immune responses. Here we report the expression, purification, and functional characterization of M. sexta HP2. The HP2 precursor is synthesized in hemocytes, fat body, integument, nerve and trachea. Its mRNA level is low in fat body of 5th instar larvae before wandering stage; abundance of the protein in hemolymph displays a similar pattern. HP2 exists as an active enzyme in plasma of the wandering larvae and pupae in the absence of an infection. HP14 cleaves proHP2 to yield active HP2. After incubating active HP2 with larval hemolymph, we detected higher levels of PO activity, i.e. an enhancement of proPO activation. HP2 cleaved proPAP2 (but not proPAP3 or proPAP1) to yield active PAP2, responsible for a major increase in IEARpNA hydrolysis. PAP2 activates proPOs in the presence of a cofactor of SPH1 and SPH2. In summary, we have identified a new member of the proPO activation system and reconstituted a pathway of HP14-HP2-PAP2-PO. Since high levels of HP2 mRNA were present in integument and active HP2 in plasma of wandering larvae, HP2 likely plays a role in cuticle melanization during pupation and protects host from microbial infection in a soil environment.

Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy.

Metastatic melanoma remains difficult to treat despite recent approvals of several new drugs. Recently we reported encouraging results of Phase I clinical trial of radiolabeled with 188Re murine monoclonal IgM 6D2 to melanin in patients with Stage III/IV melanoma. Subsequently we generated a novel murine IgG 8C3 to melanin. IgGs are more amenable to humanization and cGMP (current Good Manufacturing Practice) manufacturing than IgMs. We performed comparative structural analysis of melanin-binding IgM 6D2 and IgG 8C3. The therapeutic efficacy of 213Bi- and 188Re-labeled 8C3 and its comparison with anti-CTLA4 immunotherapy was performed in B16-F10 murine melanoma model. The primary structures of these antibodies revealed significant homology, with the CDRs containing a high percentage of positively charged amino acids. The 8C3 model has a negatively charged binding surface and significant number of aromatic residues in its H3 domain, suggesting that hydrophobic interactions contribute to the antibody-melanin interaction. Radiolabeled IgG 8C3 showed significant therapeutic efficacy in murine melanoma, safety towards healthy melanin-containing tissues and favorable comparison with the anti-CTLA4 antibody. We have demonstrated that antibody binding to melanin relies on both charge and hydrophobic interactions while the in vivo data supports further development of 8C3 IgG as radioimmunotherapy reagent for metastatic melanoma.

Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus.

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

A melanin-mediated cancer immunotherapy patch.

Melanin is capable of transforming 99.9% of the absorbed sunlight energy into heat, reducing the risk of skin cancer. We here develop a melanin-mediated cancer immunotherapy strategy through a transdermal microneedle patch. B16F10 whole tumor lysate containing melanin is loaded into polymeric microneedles that allow sustained release of the lysate upon insertion into the skin. In combination with the near-infrared light irradiation, melanin in the patch mediates the generation of heat, which further promotes tumor-antigen uptake by dendritic cells, and leads to enhanced antitumor vaccination. We found that the spatiotemporal photoresponsive immunotherapy increases infiltration of polarized T cells and local cytokine release. These immunological effects increase the survival of mice after tumor challenge and elicited antitumor effects toward established primary tumor and distant tumor. Collectively, melanin generates local heat, boosts T cell activities by transdermal vaccines, and promotes antitumor immune responses.

Synthetic melanin bound to subunit vaccine antigens significantly enhances CD8+ T-cell responses.

Cytotoxic T-lymphocytes (CTLs) play a key role in immunity against cancer; however, the induction of CTL responses with currently available vaccines remains difficult. Because several reports have suggested that pigmentation and immunity might be functionally linked, we investigated whether melanin can act as an adjuvant in vaccines. Short synthetic peptides (8-35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides. Immunization with melanin-bound peptides efficiently triggered CTL responses in mice, even against self-antigens and at a very low dose of peptides (microgram range). Immunization against a tumor antigen inhibited the growth of established tumors in mice, an effect that was abrogated by the depletion of CD8+ lymphocytes. These results demonstrate the efficacy of melanin as a vaccine adjuvant.

Suppression of shrimp melanization during white spot syndrome virus infection.

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.

A novel type of hemocytes localizing melanization with high-spreading behavior in Mythimna separata.

Lepidopteran larvae show a cellular response to invading foreign substances that are larger than hemocytes, for example, parasitoid eggs or larvae. This response is called hemocyte encapsulation and is often accompanied by phenoloxidase (PO)-catalyzed melanization. In the present study, we artificially transplanted endoparasitoid larvae and small glass fragments into the hemocoel of the common armyworm, Mythimna separata. We observed that the host larva showed a cellular response and that, 2-4 h after transplantation, melanin formation was spatially confined to the surface of the encapsulated substances. We further noted that specific morphological hemocytes surrounded by melanin formation became attached to the surface of the foreign substances. We designated these hemocytes hyperspread cells (HSCs) on the basis of their specific characteristics and circumferential spread. We confirmed the occurrence of prophenoloxidase (PPO)/phenoloxidase (PO) on the periphery of the HSCs and in the substance secreted around the HSCs by using anti-PPO antibody. We were unable to detect PPO-mRNA in HSCs by using in situ hybridization, although we showed that oenocytoids contained PPO-mRNA and PPO protein. We used light microscopy and scanning electron microscopy to discriminate five main types of circulating M. separata hemocytes. We observed that HSCs differed from plasmatocytes, but spread out well. Further, during the encapsulation process, HSCs appeared to provide a localized melanization spot on the surface of foreign invaders.

Complement-dependent cytotoxicity of an antibody to melanin in radioimmunotherapy of metastatic melanoma.

AIM: Novel treatments for metastatic melanoma are urgently needed. MATERIALS & METHODS: We developed radioimmunotherapy of metastatic melanoma using 6D2 monoclonal antibody (mAb) to melanin with encouraging therapeutic results, preclinically and in patients. RESULTS: We observed tumor suppression with the unlabeled 6D2 mAb and investigated its tumoricidal mechanisms. In melanoma tumor-bearing mice, we detected more complement-C3 deposition in the tumors from 188-rhenium-labeled 6D2 mAb-treated mice when compared with untreated controls. 6D2 and isotype-control mAb TEPC caused suppression of tumor growth in A2058 melanoma tumor-bearing mice. Tumors of mice treated with the unlabeled 6D2 mAb were infiltrated with more lymphocytes compared with controls. In vitro antibody-dependent cell-mediated cytotoxicity did not contribute to the tumor-suppressive effect of 6D2 mAb, while 6D2 mAb demonstrated a strong effect on initiating complement-dependent cytotoxicity. CONCLUSION: We concluded that 6D2 mAb mediated complement-dependent cytotoxicity, resulting in killing of the tumor cells and suppression of tumor growth. These observations will help to improve the treatment protocols of radioimmunotherapy, as well as immunotherapy.

Prophenoloxidase system and its role in shrimp immune responses against major pathogens.

The global shrimp industry still faces various serious disease-related problems that are mainly caused by pathogenic bacteria and viruses. Understanding the host defense mechanisms is likely to be beneficial in designing and implementing effective strategies to solve the current and future pathogen-related problems. Melanization, which is performed by phenoloxidase (PO) and controlled by the prophenoloxidase (proPO) activation cascade, plays an important role in the invertebrate immune system in allowing a rapid response to pathogen infection. The activation of the proPO system, by the specific recognition of microorganisms by pattern-recognition proteins (PRPs), triggers a serine proteinase cascade, eventually leading to the cleavage of the inactive proPO to the active PO that functions to produce the melanin and toxic reactive intermediates against invading pathogens. This review highlights the recent discoveries of the critical roles of the proPO system in the shrimp immune responses against major pathogens, and emphasizes the functional characterizations of four major groups of genes and proteins in the proPO cascade in penaeid shrimp, that is the PRPs, serine proteinases, proPO and inhibitors.

Increased susceptibility of melanin-concentrating hormone-deficient mice to infection with Salmonella enterica serovar Typhimurium.

Melanin-concentrating hormone (MCH) was initially identified in mammals as a hypothalamic neuropeptide regulating appetite and energy balance. However, the wide distribution of MCH receptors in peripheral tissues suggests additional functions for MCH which remain largely unknown. We have previously reported that mice lacking MCH develop attenuated intestinal inflammation when exposed to Clostridium difficile toxin A. To further characterize the role of MCH in host defense mechanisms against intestinal pathogens, Salmonella enterocolitis (using Salmonella enterica serovar Typhimurium) was induced in MCH-deficient mice and their wild-type littermates. In the absence of MCH, infected mice had increased mortality associated with higher bacterial loads in blood, liver, and spleen. Moreover, the knockout mice developed more-severe intestinal inflammation, based on epithelial damage, immune cell infiltrates, and local and systemic cytokine levels. Paradoxically, these enhanced inflammatory responses in the MCH knockout mice were associated with disproportionally lower levels of macrophages infiltrating the intestine. Hence, we investigated potential direct effects of MCH on monocyte/macrophage functions critical for defense against intestinal pathogens. Using RAW 264.7 mouse monocytic cells, which express endogenous MCH receptor, we found that treatment with MCH enhanced the phagocytic capacity of these cells. Taken together, these findings reveal a previously unappreciated role for MCH in host-bacterial interactions.

Melanoma stem cells in experimental melanoma are killed by radioimmunotherapy.

INTRODUCTION: In spite of recently approved B-RAF inhibitors and immunomodulating antibodies, metastatic melanoma has poor prognosis and novel treatments are needed. Melanoma stem cells (MSC) have been implicated in the resistance of this tumor to chemotherapy. Recently we demonstrated in a Phase I clinical trial in patients with metastatic melanoma that radioimmunotherapy (RIT) with 188-Rhenium((188)Re)-6D2 antibody to melanin was a safe and effective modality. Here we investigated the interaction of MSC with RIT as a possible mechanism for RIT efficacy. METHODS: Mice bearing A2058 melanoma xenografts were treated with either 1.5 mCi (188)Re-6D2 antibody, saline, unlabeled 6D2 antibody or (188)Re-labeled non-specific IgM. RESULTS: On Day 28 post-treatment the tumor size in the RIT group was 4-times less than in controls (P<0.001). The tumors were analyzed by immunohistochemistry and FACS for two MSC markers--chemoresistance mediator ABCB5 and H3K4 demethylase JARID1B. There were no significant differences between RIT and control groups in percentage of ABCB5 or JARID1B-positive cells in the tumor population. Our results demonstrate that unlike chemotherapy, which kills tumor cells but leaves behind MSC leading to recurrence, RIT kills MSC at the same rate as the rest of tumor cells. CONCLUSIONS: These results have two main implications for melanoma treatment and possibly other cancers. First, the susceptibility of ABCB5+ and JARID1B+cells to RIT in melanoma might be indicative of their susceptibility to antibody-targeted radiation in other cancers where they are present as well. Second, specifically targeting cancer stem cells with radiolabeled antibodies to ABCB5 or JARID1B might help to completely eradicate cancer stem cells in various cancers.

Immunity to melanin and to tyrosinase in melanoma patients, and in people with vitiligo.

BACKGROUND: The aim of this study was to determine the presence and the intensity of humoral immunity to melanoma-associated antigens: tyrosinase and melanin, in patients with melanoma, in persons with vitiligo and in control healthy people. METHODS: The study involved 63 patients with melanoma and 19 persons with vitiligo. Control group consisted up to 41 healthy volunteers. Mushroom tyrosinase and synthetic melanin were used as the antigens. RESULTS: ELISA test showed significantly (p < 0.0000004 and p < 0.04) lower levels of IgM anti-tyrosinase autoantibodies, in melanoma and vitiligo patients respectively, compared to controls.Although there was no significant difference between the levels of IgA anti-melanin autoantibodies in melanoma or vitiligo patients in comparison with controls, the enhanced concentrations of anti-melanin IgA autoantibodies were preferentially found in melanoma patients with metastatic disease. Significantly high percentage in the Fc alphaRI (CD89) positive cells was determined in melanoma patients (p < 0.002 and p < 0.008) in comparison to that found in healthy people or in patients with vitiligo, in the already mentioned order, pointing that IgA dependent cellular cytotoxicity is not important for the immune action against melanoma, even more that it is included in some immune suppression.Levels of IgG autoantibodies to mentioned antigens in melanoma patients although low were not significantly lower from controls. These findings analyzed together with the statistically significant low percentage of FcgammaRIII, (CD16) positive immunocompetent cells (p < 0.0007 and p < 0.003), which was found in patients with melanoma compared with healthy or vitiligo people respectively, and statistically significant low percentage of (CD16 + CD56+) natural killer (NK) cells (p < 0.005) found in melanoma patients in comparison to healthy controls pointed to the low probability for anti-melanoma IgG mediated, antibody mediated cellular cytotoxicity, (ADCC) and NK cytotoxicity. Moreover the ratio of the percentages of granulocytes and percentage of lymphocytes was statistically higher in patients with melanoma in relation to healthy people as well as to people with vitiligo (p < 0.0007 and p < 0.05 respectively). CONCLUSION: Autoantibodies to tyrosinase and to melanin which are found even in healthy people, point that consummation of edible mushrooms that carry the antigen tyrosinase and melanin, could influence the humoral anti-melanoma immune response.Levels of different immunoglobulin classes of anti-melanin and anti-tyrosinase antibodies varied depending on the presence and the stage of studied diseases. Besides, the statistically enhanced ratio of the percentages of granulocytes and percentage of lymphocytes, together with statistically decreased percentage of NK cells is found in analyzed melanoma patients.

Pigment-producing granulomatous myopathy in Atlantic salmon: a novel inflammatory response.

Melanin comprises a complex group of pigmented polymers whose primary function is ascribed to dermal solar protection, but may also have an interesting role in innate immunity. In ectothermic vertebrates, melanogenesis is reported in leukocyte populations, but it is not known if this occurs in connection with inflammatory reactions. Melanin accumulations in ectopic locations, in particular muscle, represent a serious quality problem in salmon production. Here, we investigated such changes for the expression of dopachrome tautomerase and tyrosinase as well as some important immune genes and pathogens. Furthermore, the nature of the pathological changes was addressed by morphological methods. Gene transcripts encoding key enzymes in melanogenesis, suggesting a de novo melanin synthesis in pigmented muscle, were found. MHC class II transcripts were up-regulated and there was no indication of bacterial or viral infection. The histological examination revealed granulomatous inflammation with distribution of MHC class II positive cells and T cells, analogous to the pattern found in mammals. Importantly, in contrast to mammals pigmented cells were contributing in the inflammation. We demonstrate that melanin production occurs in granulomatous inflammation in salmon, revealing a close and hitherto unreported link between the pigmentary and immune systems.

Detection of antibodies against Paracoccidioides brasiliensis melanin in in vitro and in vivo studies during infection.

Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.

Phagocytosis of melanized Aspergillus conidia by macrophages exerts cytoprotective effects by sustained PI3K/Akt signalling.

Host cell death is a critical component of innate immunity and often determines the progression and outcome of infections. The opportunistic human pathogen Aspergillus fumigatus can manipulate the immune system either by inducing or by inhibiting host cell apoptosis dependent on its distinct morphological form. Here, we show that conidia of Aspergillus ssp. inhibit apoptosis of macrophages induced via the intrinsic (staurosporine) and extrinsic (Fas ligand) pathway. Hence, mitochondrial cytochrome c release and caspase activation were prevented. We further found that the anti-apoptotic effect depends on both host cell de novo protein synthesis and phagocytosis of conidia by macrophages. Moreover, sustained PI3K/Akt signalling in infected cells is an important determinant to resist apoptosis. We demonstrate that pigmentless pksP mutant conidia of A. fumigatus failed to trigger protection against apoptosis and provide evidence that the sustained survival of infected macrophages depends on the presence of the grey-green conidial pigment consisting of dihydroxynaphthalene-melanin. In conclusion, we revealed a novel potential function of melanin in the pathogenesis of A. fumigatus. For the first time, we show that melanin itself is a crucial component to inhibit macrophage apoptosis which may contribute to dissemination of the fungus within the host.