Appropriate forestry practices for the management of austrian pine (Pinus nigra Arn.) plantations with understory of manna ash (Fraxinus ornus L.) in Sofia surrounding mountains

The object of the current study are Austrian pine (Pinus nigra Arn.) plantations with an understory of Manna ash (Fraxinus ornus L.), located in the mountain areas near Sofia, Bulgaria on altitude from 700 to 850 m a.s.l. The aim is to study the natural regeneration processes taking place in the plantations and to provide guidelines for their management. It has been established that their transformation into deciduous stands is a natural process that is currently ongoing. The new generation of trees consists mainly of native oak species and their natural companions. However, the Manna ash understory is overgrowing and silencing the seedlings. The amount of seedlings depends on the forest site and increases as growing conditions improve. With the increase of canopy density, understory density decreases rapidly, while the amount of seedlings does not change significantly. With the increase of stand age, the amount of seedlings increases at the expense of the understory. Increasing the height of the understory results in significant loss of seedlings. In order to create conditions for successful transformation of the pine plantations with economically valuable autochthonous species, it is recommended to manage them at a canopy density above 0.6 as long as possible. Usually, a sufficient amount of seedlings is available after the stand age of 65, when the felling for their transformation can also begin. Forest sites with better conditions have seedlings present in the entire area, while in forest sites with worse conditions their distribution is uneven. In the first years of the felling the number of required thinnings depends on the height of the understory, which should not be allowed to exceed 120 cm until the new stand forms a canopy

Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition

Antibodies classically bind antigens via their complementarity-determining regions, but an alternative mode of interaction involving V-domain framework regions has been observed for some B-cell “superantigens”. We report the first crystal structure of an antibody employing both modes of interaction simultaneously and binding two antigen molecules. This human antibody from an allergic individual binds to the grass pollen allergen Phl p 7. Not only are two allergen molecules bound to each antibody Fab, but also each allergen molecule is bound by two Fabs: one epitope is recognized classically, the other in a superantigen-like manner. A single allergen molecule thus cross-links two identical Fabs, contrary to the one-antibody/one-epitope dogma which dictates that a dimeric allergen at least is required for this to occur. Allergens trigger immediate hypersensitivity reactions by cross-linking receptor-bound IgE molecules on effector cells. We found that monomeric Phl p 7 induced degranulation of basophils sensitized solely with this monoclonal antibody expressed as an IgE, demonstrating that the dual specificity has functional consequences. The monomeric state of Phl p 7 and two structurally-related allergens was confirmed by size-exclusion chromatography and multi-angle laser light scattering, and the results were supported by degranulation studies with the related allergens, a second patient-derived allergen-specific antibody lacking the non-classical binding site, and mutagenesis of the non-classically recognized allergen epitope. This antibody dual reactivity and novel cross-linking mechanism not only has implications for understanding allergenicity and allergen potency, but importantly has broader relevance to antigen recognition by membrane immunoglobulin and cross-linking of the B-cell receptor.<br/

A tool kit for rapid cloning and expression of recombinant antibodies

Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG(1)/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG(4)/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions

A tool kit for rapid cloning and expression of recombinant antibodies

Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions

Damage caused by singing cicadas (Hemiptera: Cicadidae) in the field protective forest belts in South Dobrudzha, Bulgaria

During the period 2020-2023, strong damage caused by singing cicadas (Hemiptera: Cicadidae) were registered on ash trees (Fraxinus spp.) in the field protective forest belts (FPFBs) in South Dobrudzha, northeastern Bulgaria. Bioacoustic studies have shown that the sounds are of Cicada orni. Many exuvia of the species were also found on the trunks and branches of ash trees. On the upper shoots and petioles, numerous oviposition holes were observed, which lead to leaf fall and drying of branch tips. In different FPFBs, tree crown damage ranges from a moderate (25-60% defoliation) to a severe (over 60% defoliation) degree. The attacks were stronger on Fraxinus excelsior and F. americana compared to F. angustifolia. The cicadas affect both old trees and young ash saplings. In young plantations, other tree species (Sophora japonica, Gleditsia triacanthos) were also affected. Imaginal activity of Cicada orni was recorded in July and August, and the peak of egg hatching occurred from early August to mid-September. The high number of Cicada orni necessitates the development of measures to control the pest in the FPFBs

Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition

Antibodies classically bind antigens via their complementarity-determining regions, but an alternative mode of interaction involving V-domain framework regions has been observed for some B cell "superantigens." We report the crystal structure of an antibody employing both modes of interaction simultaneously and binding two antigen molecules. This human antibody from an allergic individual binds to the grass pollen allergen Phl p 7. Not only are two allergen molecules bound to each antibody fragment (Fab) but also each allergen molecule is bound by two Fabs: One epitope is recognized classically, the other in a superantigen-like manner. A single allergen molecule thus cross-links two identical Fabs, contrary to the one-antibody-one-epitope dogma, which dictates that a dimeric allergen at least is required for this to occur. Allergens trigger immediate hypersensitivity reactions by cross-linking receptor-bound IgE molecules on effector cells. We found that monomeric Phl p 7 induced degranulation of basophils sensitized solely with this monoclonal antibody expressed as an IgE, demonstrating that the dual specificity has functional consequences. The monomeric state of Phl p 7 and two structurally related allergens was confirmed by size-exclusion chromatography and multiangle laser light scattering, and the results were supported by degranulation studies with the related allergens, a second patient-derived allergen-specific antibody lacking the nonclassical binding site, and mutagenesis of the nonclassically recognized allergen epitope. The antibody dual reactivity and cross-linking mechanism not only have implications for understanding allergenicity and allergen potency but, importantly, also have broader relevance to antigen recognition by membrane Ig and cross-linking of the B cell receptor

IgE re-programs alternatively-activated human macrophages towards pro-inflammatory anti-tumoural states

Background Antibody Fc-driven engagement of macrophages is critical for evoking cellular activation and effector functions and influencing tumour-associated macrophage (TAM) recruitment. We previously reported that IgE class antibodies promote restriction of cancer growth in rodent models associated with significant TAM infiltration. However, the human macrophage-associated IgE-Fc Receptor (FcεR) axis remains unexplored. We investigated the effects of anti-tumour IgE stimulation on human macrophage activation. Methods Human blood monocyte-differentiated quiescent (M0), classically-(M1) and alternatively-(M2) activated macrophages were crosslinked with IgE and polyclonal antibodies to mimic immune complex formation. We examined surface marker expression, cytokine secretion, protein kinase phosphorylation and gene expression in IgE-stimulated macrophages and IgE antibody-dependent macrophage-mediated cytotoxicity (ADCC) against tumour cells. Findings A proportion (40%) of M2 and (&lt;20%) M0 and M1 macrophages expressed the high-affinity IgE receptor FcεRI. IgE crosslinking triggered upregulation of co-stimulatory CD80, increased TNFα, IFNγ, IL-1β, IL-12, IL-10, IL-13, CXCL9, CXCL11 and RANTES secretion by M0 and M2 and additionally enhanced MCP-1 by M2 macrophages. IgE-stimulated M1 macrophages retained secretion of pro-inflammatory cytokines. IgE crosslinking enhanced the FcεRI-dependent signalling pathway, including phosphorylation of the Lyn kinase, ERK1/2 and p38 in M2 macrophages and upregulated Lyn gene expression by M1 and M2 macrophages. Anti-tumour IgE engendered ADCC of cancer cells by all macrophage subsets. Interpretation IgE can engage and re-educate alternatively-activated macrophages towards pro-inflammatory phenotypes and prime all subsets to mediate anti-tumour functions. This points to IgE-mediated cascades with potential to activate immune stroma and may be significant in the clinical development of strategies targeting tumour-resident macrophages

Anti-folate receptor-α IgE but not IgG recruits macrophages to attack tumors via TNFa/MCP-1 signaling

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibodydependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen-specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fce receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG.</p