Antimicrobial activity of peach and grapevine defensins

Antimicrobial peptides (AMPs) are an important component of the innate immune system of the plants. Plant defensins are a large family of antimicrobial peptides with several interesting features, such as small dimension, high stability and broad spectrum of action. The discovery of new molecules and the study of their mechanism of action allow to consider them attractive for biotechnological applications. In this PhD thesis a defensin from Prunus persica (PpDFN1) and four novel DEFensin Like (DEFL) peptides from Vitis vinifera have been studied. In order to characterize the antimicrobial activity of these molecules, the recombinant mature peptides have been expressed in Escherichia coli and purified to homogeneity by chromatography techniques. PpDFN1 is able to inhibit the growth of B. cinerea, P. expansum and M. laxa with different intensity. The recombinant peptide is capable of membrane permeabilization as demonstrated by SYTOX green fluorescence uptake in treated mycelia. Its interaction with membranes containing sphingolipid species has been shown by artificial lipid monolayers. Furthermore, PpDFN1 displays stronger interaction with monolayers composed by lipids extracted from sensitive fungi with the highest interaction against P. expansum, the most sensitive fungi to PpDFN1 action. DEFL 13, a defensin from grapevine, resulted the strongest antibotrytis peptides. It is electrostatically attracted to the fungal membranes as shown by the antagonist effect of the cations and is able to membrane permeabilization in B. cinerea hyphae. DEFL 13 is internalized in fungal cells and leads to fungal death by activation of some signaling pathways as demonstrated by screening of a mutant collection of B. cinerea

In-vitro synthesis and reconstitution of cytochrome bo 3 ubiquinol oxidase in artificial membranes

Forschung über Membranenproteine stellt strenge Hindernisse, seit ruhigem gerade wenige Beispiele der Membranenproteinsorten sind gekennzeichnet worden in den verwendbaren experimentellen Plattformen gegenüber. Die Hauptherausforderung ist, ihre ausgezeichnete entworfene strukturelle Vollständigkeit zu konservieren, während die Ausdruck-, Lokalisierungs- und Wiederherstellungprozesse auftreten. In-vitro übersetzungssysteme können Vorteile über auf Zellenbasisgenausdruck zum Beispiel haben, wenn das über-ausgedrückte Produkt zur Wirtszelle giftig ist oder wenn fehlende Pfosten-Übersetzungsänderung in den bakteriellen Ausdrucksystemen die Funktionalität der Säugetier- Proteine oder Mangel an vorhandenem Membranenraum verdirbt, Funktionsausdruck verbieten.rn Der Nachahmer von biologische Membranen wie feste gestützte Lipidmembranen sind als Plattform am meisten benutzt, Proteinmembraneninteraktionen nachzuforschen. Wir sind in der Lage, Membranenproteinsorte, da wir eine Plattform für Membranenproteinsynthese vorstellen, nämlich die in-vitrosynthese der Membranenproteine in ein Peptid gestütztes Membranensystem zu adressieren. Die Wiederherstellung der Membranenproteine in den Lipid bilayers resultiert im Allgemeinen mit verschiedenen Proteinanpassungen. Als Alternative erforschen wir dieses System zum ersten Mal, um genaueres Modell zu den zellularen Membranen zu verursachen und ihre Funktion, wie Proteineinfügung, Proteinfunktion und Ligandinteraktionen nachzuahmen.rn In dieser Arbeit ist unser Ziel, komplizierte Transmembraneproteine, wie des Cytochrome bo3-ubiquinol Oxydase (Cyt-bo3) direkt innerhalb der biomimetic vorbildlichen Membrane zu synthetisieren. In unserem System wird festes gestütztes tBLM wie, P19/DMPE/PC als Plattform benutzt. Dieses künstliche Membranensystem mimiks die amphiphile Architektur eines Zelle-abgeleiteten Membranensystems.rn

An interferon regulatory factor element controls the major immune modulator of murine cytomegalovirus

Immune modulation by herpesviruses, such as cytomegalovirus, is critical for the establishment of acute and persistent infection confronting a vigorous antiviral immune response of the host. Therefore, the action of immune-modulatory proteins has long been the subject of research, with the final goal to identify new strategies for antiviral therapy.rnIn the case of murine cytomegalovirus (mCMV), the viral m152 protein has been identified to play a major role in targeting components of both the innate and the adaptive immune system in terms of infected host-cell recognition in the effector phase of the antiviral immune response. On the one hand, it inhibits cell surface expression of RAE-1 and thereby prevents ligation of the activating natural killer (NK)-cell receptor NKG2D. On the other hand, it decreases cell surface expression of peptide-loaded MHC class I molecules thereby preventing antigen presentation to CD8 T cells. Ultimately, the outcome of CMV infection is determined by the interplay between viral and cellular factors.rnIn this context, the work presented here has revealed a novel and intriguing connection between viral m152 and cellular interferon (IFN), a key cytokine of the immune system: rnthe m152 promoter region contains an interferon regulatory factor element (IRFE) perfectly matching the consensus sequence of cellular IRFEs.rnThe biological relevance of this regulatory element was first suggested by sequence comparisons revealing its evolutionary conservation among various established laboratory strains of mCMV and more recent low-passage wild-derived virus isolates. Moreover, search of the mCMV genome revealed only three IRFE sites in the complete sequence. Importantly, the functionality of the IRFE in the m152 promoter was confirmed with the use of a mutant virus, representing a functional deletion of the IRFE, and its corresponding revertant virus. In particular, m152 gene expression was found to be inhibited in an IRFE-dependent manner in infected cells. Essentially, this inhibition proved to have a severe impact on the immune-modulatory function of m152, first demonstrated by a restored direct antigen presentation on infected cells for CD8 T-cell activation. Even more importantly, this effect of IRFE-mediated IFN signaling was validated in vivo by showing that the protective antiviral capacity of adoptively-transferred, antigen-specific CD8 T cells is also significantly restored by the IRFE-dependent inhibition of m152. Somewhat curious and surprising, the decrease in m152 protein simultaneously prevented an enhanced activation of NK cells in acute-infected mice, apparently independent of the RAE-1/NKG2D ligand/receptor interaction but rather due to reduced ‘missing-self’ recognition.rnTaken together, this work presents a so far unknown mechanism of IFN signaling to control mCMV immune modulation in acute infection.rnrn

Transport of nanoparticles into polymersomes : a minimal model system of particles passage through biological membranes

This thesis focuses on the design and characterization of a novel, artificial minimal model membrane system with chosen physical parameters to mimic a nanoparticle uptake process driven exclusively by adhesion and softness of the bilayer. The realization is based on polymersomes composed of poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PMDS-b-PMOXA) and nanoscopic colloidal particles (polystyrene, silica), and the utilization of powerful characterization techniques. rnPDMS-b-PMOXA polymersomes with a radius, Rh ~100 nm, a size polydispersity, PD = 1.1 and a membrane thickness, h = 16 nm, were prepared using the film rehydratation method. Due to the suitable mechanical properties (Young’s modulus of ~17 MPa and a bending modulus of ~7⋅10-8 J) along with the long-term stability and the modifiability, these kind of polymersomes can be used as model membranes to study physical and physicochemical aspects of transmembrane transport of nanoparticles. A combination of photon (PCS) and fluorescence (FCS) correlation spectroscopies optimizes species selectivity, necessary for a unique internalization study encompassing two main efforts. rnFor the proof of concepts, the first effort focused on the interaction of nanoparticles (Rh NP SiO2 = 14 nm, Rh NP PS = 16 nm; cNP = 0.1 gL-1) and polymersomes (Rh P = 112 nm; cP = 0.045 gL-1) with fixed size and concentration. Identification of a modified form factor of the polymersome entities, selectively seen in the PCS experiment, enabled a precise monitor and quantitative description of the incorporation process. Combining PCS and FCS led to the estimation of the incorporated particles per polymersome (about 8 in the examined system) and the development of an appropriate methodology for the kinetics and dynamics of the internalization process. rnThe second effort aimed at the establishment of the necessary phenomenology to facilitate comparison with theories. The size and concentration of the nanoparticles were chosen as the most important system variables (Rh NP = 14 - 57 nm; cNP = 0.05 - 0.2 gL-1). It was revealed that the incorporation process could be controlled to a significant extent by changing the nanoparticles size and concentration. Average number of 7 up to 11 NPs with Rh NP = 14 nm and 3 up to 6 NPs with Rh NP = 25 nm can be internalized into the present polymersomes by changing initial nanoparticles concentration in the range 0.1- 0.2 gL-1. Rapid internalization of the particles by polymersomes is observed only above a critical threshold particles concentration, dependent on the nanoparticle size. rnWith regard possible pathways for the particle uptake, cryogenic transmission electron microscopy (cryo-TEM) has revealed two different incorporation mechanisms depending on the size of the involved nanoparticles: cooperative incorporation of nanoparticles groups or single nanoparticles incorporation. Conditions for nanoparticle uptake and controlled filling of polymersomes were presented. rnIn the framework of this thesis, the experimental observation of transmembrane transport of spherical PS and SiO2 NPs into polymersomes via an internalization process was reported and examined quantitatively for the first time. rnIn a summary the work performed in frames of this thesis might have significant impact on cell model systems’ development and thus improved understanding of transmembrane transport processes. The present experimental findings help create the missing phenomenology necessary for a detailed understanding of a phenomenon with great relevance in transmembrane transport. The fact that transmembrane transport of nanoparticles can be performed by artificial model system without any additional stimuli has a fundamental impact on the understanding, not only of the nanoparticle invagination process but also of the interaction of nanoparticles with biological as well as polymeric membranes. rn

Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA

Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.

Immune adaptations that maintain homeostasis with the intestinal microbiota

Humans harbour nearly 100 trillion intestinal bacteria that are essential for health. Millions of years of co-evolution have moulded this human-microorganism interaction into a symbiotic relationship in which gut bacteria make essential contributions to human nutrient metabolism and in return occupy a nutrient-rich environment. Although intestinal microorganisms carry out essential functions for their hosts, they pose a constant threat of invasion owing to their sheer numbers and the large intestinal surface area. In this Review, we discuss the unique adaptations of the intestinal immune system that maintain homeostatic interactions with a diverse resident microbiota.

Fas-activated serine/threonine phosphoprotein promotes immune-mediated pulmonary inflammation

We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST(-/-)) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST(-/-) mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST(-/-) mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-alpha and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST(-/-) neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.

Immune pathomechanism of drug hypersensitivity reactions

Drug hypersensitivity research has progressed enormously in recent years, and a greater understanding of mechanisms has contributed to improved drug safety. Progress has been made in genetics, enabling personalized medicine for certain drugs, and in understanding drug interactions with the immune system. In a recent meeting in Rome, the clinical, chemical, pharmacologic, immunologic, and genetic aspects of drug hypersensitivity were discussed, and certain aspects are briefly summarized here. Small chemicals, including drugs, can induce immune reactions by binding as a hapten to a carrier protein. Park (Liverpool, England) demonstrated (1) that drug haptens bind to protein in patients in a highly restricted manner and (2) that irreversibly modified carrier proteins are able to stimulate CD4(+) and CD8(+) T cells from hypersensitive patients. Drug haptens might also stimulate cells of the innate immune system, in particular dendritic cells, and thus give rise to a complex and complete immune reaction. Many drugs do not have hapten-like characteristics but might gain them on metabolism (so-called prohaptens). The group of Naisbitt found that the stimulation of dendritic cells and T cells can occur as a consequence of the transformation of a prohapten to a hapten in antigen-presenting cells and as such explain the immune-stimulatory capacity of prohaptens. The striking association between HLA-B alleles and the development of certain drug reactions was discussed in detail. Mallal (Perth, Australia) elegantly described a highly restricted HLA-B∗5701-specific T-cell response in abacavir-hypersensitive patients and healthy volunteers expressing HLA-B∗5701 but not closely related alleles. Expression of HLA-B∗1502 is a marker known to be necessary but not sufficient to predict carbamazepine-induced Stevens-Johnson syndrome/toxic epidermal necrolysis in Han Chinese. The group of Chen and Hong (Taiwan) described the possible "missing link" because they showed that the presence of certain T-cell receptor (TCR) clonotypes was necessary to elicit T-cell responses to carbamazepine. The role of TCRs in drug binding was also emphasized by Pichler (Bern, Switzerland). Following up on their "pharmacological interactions of drugs with immune receptors" concept (p-i concept), namely that drugs can bind directly to TCRs, MHC molecules, or both and thereby stimulate T cells, they looked for drug-binding sites for the drug sulfamethoxazole in drug-specific TCRs: modeling revealed up to 7 binding sites on the CDR3 and CDR2 regions of TCR Vα and Vβ. Among many other presentations, the important role of regulatory T cells in drug hypersensitivity was addressed.

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 mug of LPS or 20 mug of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12h relative to the challenge to measure mRNA expression of tumor necrosis factor-alpha (TNFalpha), IL-1beta, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFalpha. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFalpha in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFalpha, IL-1beta, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.

[Complement--a phylogenetically old system as a new player in the development of atherosclerosis]

Atherosclerotic diseases such as coronary artery disease and ischaemic stroke are caused by chronic inflammation in arterial vessel walls. The complement system is part of the innate immune system. It is involved in many processes contributing to onset and development of atherosclerotic plaques up to the final stage of acute thrombotic events. This is due to its prominent role in inflammatory processes. In addition, there is increasing evidence that interactions between complement and coagulation provide a link between inflammation and thrombosis. On the other hand, the complement system also has an atheroprotective function through the clearance of apoptotic material. The knowledge of these complex mechanisms will become increasingly important, also for clinicians, since it may lead to novel therapeutic and diagnostic options. Therapies targeting the complement system have the potential to reduce tissue damage caused by acute ischaemic events. Whether early anti-inflammatory and anti-complement therapy may be able to prevent atherosclerosis, remains a hot topic for research.

Pharmacological interaction of drugs with immune receptors: the p-i concept

Drug-induced hypersensitivity reactions have been explained by the hapten concept, according to which a small chemical compound is too small to be recognized by the immune system. Only after covalently binding to an endogenous protein the immune system reacts to this so called hapten-carrier complex, as the larger molecule (protein) is modified, and thus immunogenic for B and T cells. Consequently, a B and T cell immune response might develop to the drug with very heterogeneous clinical manifestations. In recent years, however, evidence has become stronger that not all drugs need to bind covalently to the MHC-peptide complex in order to trigger an immune response. Rather, some drugs may bind directly and reversibly to immune receptors like the major histocompatibility complex (MHC) or the T cell receptor (TCR), thereby stimulating the cells similar to a pharmacological activation of other receptors. This concept has been termed pharmacological interaction with immune receptors the (p-i) concept. While the exact mechanism is still a matter of debate, non-covalent drug presentation clearly leads to the activation of drug-specific T cells as documented for various drugs (lidocaine, sulfamethoxazole (SMX), lamotrigine, carbamazepine, p-phenylendiamine, etc.). In some patients with drug hypersensitivity, such a response may occur within hours even upon the first exposure to the drug. Thus, the reaction to the drug may not be due to a classical, primary response, but rather be mediated by stimulating existing, pre-activated, peptide-specific T cells that are cross specific for the drug. In this way, certain drugs may circumvent the checkpoints for immune activation imposed by the classical antigen processing and presentation mechanisms, which may help to explain the peculiar nature of many drug hypersensitivity reactions.

Stress-related reduction in personal mastery is associated with reduced immune cell beta2-adrenergic receptor sensitivity

Background: A growing body of literature suggests that caregiving burden is associated with impaired immune system functioning, which may contribute to elevated morbidity and mortality risk among dementia caregivers. However, potential mechanisms linking these relationships are not well understood. The purpose of this study was to investigate whether stress-related experience of depressive symptoms and reductions in personal mastery were related to alterations in ss2-adrenergic receptor sensitivity.Methods: Spousal Alzheimer's caregivers (N = 106) completed measures assessing the extent to which they felt overloaded by their caregiving responsibilities, experienced depressive symptoms, and believed their life circumstances were under their control. We hypothesized that caregivers reporting elevated stress would report increased depressive symptoms and reduced mastery, which in turn would be associated with reduced ss2- adrenergic receptor sensitivity on peripheral blood mononuclear cells (PBMC), as assessed by in vitro isoproterenol stimulation.Results: Regression analyses indicated that overload was negatively associated with mastery (beta = -0.36, p = 0.001) and receptor sensitivity (beta = -0.24, p = 0.030), whereas mastery was positively associated with receptor sensitivity (beta = 0.29, p = 0.005). Finally, the relationship between overload and receptor sensitivity diminshed upon simultaneous entry of mastery. Sobel's test confirmed that mastery significantly mediated some of the relationship between overload and receptor sensitivity (z = -2.02, p = 0.044).Conclusions: These results suggest that a reduced sense of mastery may help explain the association between caregiving burden and reduced immune cell ss2-receptor sensitivity.

Cellular immune response, stress resistance and competitiveness in nestling great tits in relation to maternally transmitted carotenoids

1. Egg yolks contain carotenoids that protect biological molecules against free-radical damage and promote maturation of the immune system. Availability of carotenoids to birds is often limited. Trade-offs can thus arise in the allocation of carotenoids to different physiological functions, and mothers may influence the immunocompetence of nestlings by modulating the transfer of carotenoid to the yolk.;2. In the great tit Parus major, we experimentally manipulated the dietary supply of carotenoid to mothers, and partially cross-fostered hatchlings to investigate the effect of an increased availability of carotenoids during egg laying on immunocompetence of nestlings.;3. In addition, we infested half of the nests with hen fleas Ceratophyllus gallinae to investigate the relationship between carotenoid availability, resistance to ectoparasites and immunocompetence.;4. We found that the procedure of cross-fostering can reduce the immune response of nestlings, but this effect can be compensated by the maternally transferred carotenoids. Cross-fostered nestlings of carotenoid-supplemented females show a similar immune response to non-cross-fostered nestlings, while cross-fostered nestlings of control females mounted a weaker cell-mediated immune response. This suggests that yolk carotenoids may help nestlings to cope with stress, for example the one generated by cross-fostering and/or they may enhance nestling competitiveness.;5. There was no statistically significant interaction between parasite and carotenoid treatments, as would be expected if carotenoids helped nestlings to fight parasites. Under parasite pressure, however, lighter nestlings raised a lower immune response, while the immune response was only weakly correlated with body mass in uninfested nests.

Carotenoid-based plumage colors and immune function: Is there a trade-off for rare carotenoids?

Theory suggests that carotenoid-based signals are used in animal communication because they contain specific information about parasite resistance or immunocompetence. This implies that honesty of carotenoid-based signals is maintained by a trade-off between pigmentation and immune function for carotenoids, assuming that the carotenoids used for coloration are also immunoenhancing. We tested this hypothesis by altering the diets of nestling great tits (Paris major) with supplementary beadlets containing the carotenoids that are naturally ingested with food or beadlets containing the carotenoids that are incorporated into the feathers; a control group received beadlets containing no carotenoids. We simultaneously immune challenged half of the nestlings of each supplementation group, using a two-factorial design. Activatior of the immune system led to reduced color expression. However, only nestlings fed with the naturally ingested carotenoids and not with the carotenoids deposited in the feathers showed an increased cellular immune response. This shows that the carotenoids used for ornamentation do not promote the immune function, which conflicts with the trade-off hypothesis. Our results indicate that honesty of carotenoid-based signals is maintained by an individual's physiological limitation to absorb and/or transport carotenoids and by access to carotenoids, indicating that preferences for carotenoid-based traits in sexual selection or parent-offspring interactions select for competitive individuals, rather than specifically for immune function.

Time course of biological activity in fresh murine osteochondral allografts paralleled to the recipient's immune response

The use of fresh osteochondral allografts is a popular approach to treat articular cartilage lesions. Immunological reactions of the recipient elicited by the allograft's osseous portion, however, frequently result in their deterioration. So far, little emphasis has been put on describing morphology and biological activity in fresh allografts and paralleling these to the immunological processes triggered in the host. Therefore, in the present study murine neonatal femora, serving as osteochondral grafts, were transplanted as fresh isografts (controls) or allografts (the latter in non- or presensitized mice) and retrieved after 2, 5, 10, and 20 days. It was shown that (1) in isografts active bone cells (osteoblasts, osteoclasts) were present, the bone marrow was repopulated with hematopoietic cells, the diaphysis increased in length, and no specific immunological reaction by the recipient was evoked. (2) Allografts transplanted into nonsensitized hosts initially appeared similar as isografts, but activated T lymphocytes at the transplantation site preceded loss of active bone cells within the graft and development of fibrosis within the marrow cavity. (3) In allografts transplanted into presensitized recipients, severe deterioration of the graft was observed with very few active bone cells, accompanied by an invasion of T lymphocytes and fibrosis in the marrow cavity already in early stages. Similar to vital organ transplantation, the function of cells within osteochondral allografts is severely impaired after being recognized by the immune system. Therefore, emphasis has to be placed on the development of procedures preserving cartilage biology while reducing the antigenicity of the allograft's osseous portion.