Spontaneous repair of full-thickness defects of articular cartilage in a goat model. A preliminary study.

BACKGROUND: Full-thickness defects measuring 3 mm in diameter have been commonly used in studies of rabbits to evaluate new procedures designed to improve the quality of articular cartilage repair. These defects initially heal spontaneously. However, little information is available on the characteristics of repair of larger defects. The objective of the present study was to define the characteristics of repair of 6-mm full-thickness osteochondral defects in the adult Spanish goat. METHODS: Full-thickness osteochondral defects measuring 6 x 6 mm were created in the medial femoral condyle of the knee joint of adult female Spanish goats. The untreated defects were allowed to heal spontaneously. The knee joints were removed, and the defects were examined at ten time-intervals, ranging from time zero (immediately after creation of the defect) to one year postoperatively. The defects were examined grossly, microradiographically, histologically, and with magnetic resonance imaging and computed tomography. RESULTS: The 6-mm osteochondral defects did not heal. Moreover, heretofore undescribed progressive, deleterious changes occurred in the osseous walls of the defect and the articular cartilage surrounding the defect. These changes resulted in a progressive increase in the size of the defect, the formation of a large cavitary lesion, and the collapse of both the surrounding subchondral bone and the articular cartilage into the periphery of the defect. Resorption of the osseous walls of the defect was first noted by one week, and it was associated with extensive osteoclastic activity in the trabecular bone of the walls of the defect. Flattening and deformation of the articular cartilage at the edges of the defect was also observed at this time. By twelve weeks, bone resorption had transformed the surgically created defect into a larger cavitary lesion, and the articular cartilage and subchondral bone surrounding the defect had collapsed into the periphery of the defect. By twenty-six weeks, bone resorption had ceased and the osseous walls of the lesion had become sclerotic. The cavitary lesion did not become filled in with fibrocartilage. Instead, a cystic lesion was found in the center of most of the cavitary lesions. Only a thin layer of fibrocartilage was present on the sclerotic osseous walls of the defect. Specimens examined at one year postoperatively showed similar characteristics. CONCLUSIONS: Full-thickness osteochondral defects, measuring 6 mm in both diameter and depth, that are created in the medial femoral condyle of the knee joint of adult Spanish goats do not heal spontaneously. Instead, they undergo progressive changes resulting in resorption of the osseous walls of the defect, the formation of a large cavitary lesion, and the collapse of the surrounding articular cartilage and subchondral bone. CLINICAL RELEVANCE: As surgeons apply new reparative procedures to larger areas of full-thickness articular cartilage loss, we believe that it is important to consider the potential deleterious effects of a "zone of influence" secondary to the creation of a large defect in the subchondral bone. When biologic and synthetic matrices with or without cells or bioactive factors are placed into surgically created osseous defects, the osseous walls serve as shoulders to protect and stabilize the preliminary repair process. It is important to protect the repair process until biologic incorporation occurs and the chondrogenic switch turns the cells on to synthesize an articular-cartilage-like matrix. It takes a varying period of time to fill a large, surgically created bone defect underlying a chondral surface. The repair of such a defect requires bone synthesis and the reestablishment of a subchondral plate with a tidemark transition to the new overlying articular surface. The prevention of secondary changes in the surrounding bone and articular cartilage and the durability of the new reparative tissue making up the articulating surface are issues that must be addressed in future studies.

Migration of polyethylene particles around stable implants in an animal model.

The aim of this study was to test the hypothesis that a tight seal between bone and implant will eliminate the avenue of particle migration around stable implants. Three types of implants were used in rabbits (polished press-fit Ti-6Al-4V or plasma-sprayed hydroxyapatite [HA]-coated Ti-6Al-4V) or doughy stage polymethyl methacrylate (PMMA). Implants were placed in the condylar notch. Each animal received an intra-articular injection of high density polyethylene (PE) particles (10(8) in 0.4 mL; mean size 4.7 microns) at 4 and 6 weeks postoperatively. Eight weeks postoperatively, peri-implant tissues were examined for PE particles and osteolysis. In all cases, intracellular PE particles were seen at the bone-implant interface and within marrow. No osteolysis was observed. Bone apposition was determined by computerized image analysis. There was no significant difference in the percentage of bone apposition (+/- SD) among the three groups of implants: Ti-6Al-4V (68% +/- 19%), HA-coated Ti-6Al-4V (70% +/- 10%), and PMMA (59% +/- 12%). These results indicate that a polished Ti-6Al-4V surface is as effective as PMMA or HA coating in limiting migration of PE particles around stable osseointegrated implants in rabbits.

Pathogenicity of Theileria parva is influenced by the host cell type infected by the parasite.

Theileria parva has been shown to infect and transform B cells and T cells at similar frequencies in vitro. However, the majority of parasitized cells in the tissues of infected cattle are alpha/beta T cells. The aim of this study was to determine whether the cell type infected with T. parva influenced the pathogenicity of the parasite. The initial approach, which involved inoculation of cattle with autologous cloned cell lines of different phenotypes, failed to resolve the issue, because of prolonged period of culture required to clone and characterize the cell lines resulted in attenuation of the cells. As an alternative approach, cattle were inoculated with purified populations of autologous cells that had been incubated in vitro with T. parva sporozoites for 48 h. As few as 3 x 10(4) peripheral blood mononuclear cells (PBMC) treated in this way were found to produce severe clinical reactions with high levels of parasitosis. Infections of similar severity were produced with purified populations of CD2+, CD4+, and CD8+ T cells. By contrast, infected B cells gave rise to mild self-limiting infections even when administered at a 10-fold-higher dose. In animals that received infected CD4+ or CD8+ T cells, the parasitized cells in the lymph nodes on day 11 of infection were all within the CD4+ and CD8+ populations, respectively, indicating that there had been minimal transfer of the parasite between cell types. Phenotypic analyses of cultures of PBMC infected in vitro with saturating concentrations of sporozoites revealed that parasitized B cells were abundant in the cultures after 1 week but were subsequently overgrown by T cells. The results of these experiments indicate that the cell type infected by T. parva influences the pathogenicity of the parasite.

Antigen-driven B cell differentiation in vivo.

The secretion of specific antibodies and the development of somatically mutated memory B cells in germinal centers are consequences of T cell-dependent challenge with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). Using six-parameter flow cytometry and single cell molecular analysis we can directly monitor the extent of somatic hypermutation in individual responsive (isotype switched) antigen-specific B cells. The current study provides a direct quantitative assessment of recruitment into the antibody-secreting compartment on the one hand, and the germinal center pathway to memory on the other. Cellular expansion in both compartments is exponential and independent during the first week after challenge. The first evidence of somatic mutation, towards the end of the first week, was restricted to the germinal center pathway. Furthermore, germinal center cells express a significantly shorter third hypervariable region (CDR3), even when unmutated, than their antibody-secreting counterparts, suggesting a secondary selection event may occur at the bifurcation of these two pathways in vivo. By the end of the second week, the majority of mutated clones express a shorter CDR3 and affinity-increasing mutations as evidence of further selection after somatic mutation. These data provide evidence for substantial proliferation within germinal centers before the initiation of somatic mutation and the subsequent selection of a significant frequency of mutated clonotypes into the memory compartment.

Functional and molecular characterization of single, (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific, IgG1+ B cells from antibody-secreting and memory B cell pathways in the C57BL/6 immune response to NP.

We have used multiparameter flow cytometry to identify a population of IgG1+ IgM- antigen-specific B cells which emerges in spleens of C57BL/6 mice following immunization with the hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP). Characterization of the specificities of IgG1 antibodies produced by single, sorted IgG1+ NP+ cells in both Elispot assays and in microcultures containing lipopolysaccharide, interleukin (IL)-2, IL-4 and IL-5 indicates that the splenic IgG1+ NP+ B cell population includes both IgG1 anti-NP antibody-secreting cells and non-secreting, IgG1+ memory B cells. Each functionally discrete population of IgG1+ B cells expresses a distinctive surface phenotype defined by a wide range of B cell markers. In particular, antibody-secreting, IgG1+ cells were uniquely identified by co-expression of the matrix receptor, syndecan. The NP-specific B cell population emerging in the day 7 primary response was assessed for clonotypic diversity by amplification and direct sequencing of the rearranged V186.2 heavy chain variable region gene expressed by single, ex vivo IgG1+ NP+ lambda+ B cells. Memory B cell clones, distinguished by junctional diversity, carried either no mutation or a single mutation within rearranged V186.2, suggesting isolation of these cells at the onset of the hypermutation mechanism. This novel approach, therefore, allows the direct and unambiguous identification and characterization of individual B cell clonotypes during their initial selection and activation in antibody responses in vivo.

Molecular characterization of single memory B cells.

Primary antigenic exposure results in an initial antibody response and the T cell-dependent induction of B-cell memory. Memory B-cell differentiation is characterized by somatic hypermutation in antibody variable region genes (V) and selection of B cells expressing high-affinity variants of this antigen receptor. Despite our current understanding of B-cell memory, the origin of memory B cells and the regulation of their differentiation remain elusive. This is largely due to the difficulties in observing and purifying this minor component of the immunized spleen. Further, molecular characterization of memory B cells requires hybridoma formation which restricts analyses to only those clones capable of fusion and does not allow isolation of cells in a normal physiological state. We have therefore developed a unique system which allows isolation and unambiguous enumeration of IgG1+ memory B cells, based on six-parameter flow cytometry, secretion of antibody in clonal cultures and analysis of clonally expressed V genes using the polymerase chain reaction. Here we report that single IgG1+ antigen-binding B cells from an early secondary immune response proliferate in lipopolysaccharide-driven microcultures and produce antigen-specific IgG1 antibodies. Individual B-cell clones in these cultures express somatically mutated heavy chain V genes, confirming their designation as memory B cells. Although isolated memory B cells undergo extensive proliferation in vitro, V gene sequence analysis of their individual progeny shows that further hypermutation does not occur.

Sensitivity to titanium. A cause of implant failure?

Tissues from five patients who underwent revision operations for failed total hip replacements were found to contain large quantities of particulate titanium. In four cases this metal must have come from titanium alloy screws used to fix the acetabular component; in the fifth case it may also have originated from a titanium alloy femoral head. Monoclonal antibody labelling showed abundant macrophages and T-lymphocytes, in the absence of B-lymphocytes, suggesting sensitisation to titanium. Skin patch testing with dilute solutions of titanium salts gave negative results in all five patients. However, two of them had a positive skin test to a titanium-containing ointment.

An evolutionarily-conserved role for murine Ly-1 B cells in protection against bacterial infections.

The murine Ly-1 B cell lineage, although comprising only a minority of peripheral IgM+ B cells, secretes a major proportion of the IgM antibodies occurring naturally in serum. Ly-1 B cells also seed a large number of IgA+ plasma cells to the gut walls, thereby contributing significantly to production of natural IgA antibodies in response to chronic stimulation by the normal gut flora. Apart from these naturally-produced antibodies, Ly-1 B cells also produce specific antibodies following deliberate immunisation with the bacterial cell wall antigens, phosphorylcholine and dextran. The inability of the X-linked immunodeficient CBA/N mice to produce antibody responses to these two antigens is overcome by reconstitution with normal Ly-1 B cells from the parental CBA strain. Ly-1 B cells therefore appear to play a dominant role in natural immunity and protection against bacterial infections. The compartmentalisation of development and function within murine B cells is suggestive of an evolutionary structuring of the murine immune system, with Ly-1 B cells representing a conserved, primitive B cell lineage and retaining key, associated functions.

The rat B cell system: the anatomical localization of flow cytometry-defined B cell subpopulations.

Two-color flow cytometrical (FCM) analysis of rat peripheral lymphoid organs shows two distinct IgM/IgD-defined B cell subpopulations, similar to those of the mouse: a major population of cells expressing little IgM and high levels of IgD (population I) and a minor population of cells expressing high levels of IgM but little IgD (population III). In peripheral lymphoid organs population III cells are mainly found in spleen where they represent about 25% of the B cells; population III cells are almost absent from lymph nodes and Peyer's patches. In adult bone marrow and in neonatal spleen the majority of IgM/IgD-defined B cells (greater than 70%) are population III cells, similar to what is observed in the mouse. In contrast with mice, only a low proportion of the cells (1%) recovered from the peritoneal cavity are B cells, and most of them belong to population I. Previously defined monoclonal antibodies (HIS22 and HIS24) to B cell forms of the leukocyte common antigen (CD45R) in combination with staining for surface IgM and surface IgD demonstrates a further heterogeneity of rat B cells by three-color FCM analyses. HIS22 labels most population I cells; population III cells and a small subset (about one third) of population I express only very low levels of the HIS22 determinant. HIS24 reacts with population I cells and subdivides population III into two subsets: about one third of splenic population III cells are brightly stained with this antibody whereas fluorescence of the remaining two-thirds is lower. The HIS24bright population III cells likely are newly formed B cells since cells with this phenotype are the predominant surface Ig population found in adult bone marrow and neonatal spleen. In tissue sections of lymphoid organs, HIS22- and HIS24-positive cells are mainly found in lymphoid follicles; splenic marginal zones are almost unstained. Combining immunohistological analysis with the FCM data, we therefore conclude that the small follicular B cells are in population I and marginal zone B cells are found in the HIS24dull population III. The in situ localization of HIS24bright population III cells and the HIS22dull population I cells is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)

The peritoneal Ly-1 (CD5) B cell repertoire is unique among murine B cell repertoires.

Ly-1 (CD5) B cells and conventional B cells represent two distinct lineages of murine B cells which are distinguishable by expression of surface molecules, organ location, ontogeny and development and antibody production in vivo. In order to assess whether the different developmental pathways of Ly-1 B cells and conventional B cells result in different antibody repertoires, we have used limiting dilution analyses to determine frequencies of B cells making antibodies capable of binding to a range of antigens including haptens, proteins, bacterial polysaccharides and bromelain-treated mouse red blood cells. Starting populations of B cells were purified from spleen, peritoneum and bone marrow of adult BALB/c mice or from spleens of newborn mice by use of the fluorescence-activated cell sorter. The peritoneal Ly-1 B cell repertoire was found to be different from that of conventional B cells, with between 5- and 100-fold higher frequencies of clones producing IgM antibodies capable of binding to the antigens tested. However, when tested, the majority of Ly-1 B cell anti-haptenic antibodies did not show the high affinity binding or fine specificity characteristics of specific antibodies elicited in immune responses in vivo. The high frequencies of antigen-reactive antibodies within the Ly-1 B repertoire are most likely explained by the presence of clones secreting low-affinity or multireactive antibodies. The Ly-1 B cell repertoire is not mirrored in repertoires from either newborn B cells or virgin B cells in adult bone marrow. Therefore, either Ly-1 B cells develop from distinct precursors with intrinsically different mechanisms of V gene usage and recombination, or newly formed Ly-1 B are heavily selected on specificity for entry into this peritoneal lineage. If the second alternative is true, bacterial antigens in the gut are not required for selection of this unique repertoire, as Ly-1 B cells in germ-free mice also show the multireactive repertoire characteristic of this B cell lineage in normal mice.

In vivo expression of interleukin 5 induces an eosinophilia and expanded Ly-1B lineage populations.

In vitro, murine interleukin 5 (IL-5) acts as a colony-stimulating factor for eosinophils and induces B cells to proliferate and secrete antibody. In order to assess the biological effects of IL-5 in vivo, we transplanted lethally irradiated mice with bone marrow cells infected with a recombinant retrovirus bearing the IL-5 coding sequence. Within 2 weeks the peripheral blood of recipient mice exhibited a marked eosinophilia which persisted for at least 12 months, and an excess number of eosinophils was also evident in the bone marrow, spleen, liver, lung, and gut. Although no changes could be detected in the conventional B lymphocyte population, the peritoneum was replete with B cells characteristic of the Ly-1 lineage. Despite these expanded cell populations, mice remained healthy for 12 months after transplantation. These results suggest that IL-5 acts primarily on eosinophils and B cells of the Ly-1 lineage and that persistent overproduction of these cell types is not pathogenic.

The use of foetal calf serum as a blocking agent in double immunoenzyme-cytochemistry labelling with mouse monoclonal antibodies.

Double immunoenzyme labelling has progressed in the past few years to double labelling using monoclonal antibodies (MAB) raised in the same species. It presents the problem of cross-reaction between the primary antibodies and the enzyme-labelled secondary antibodies as well as that of excessively high background staining. These difficulties have been overcome in part by using normal mouse serum as a blocking agent. However, differentiation of the resulting colour reactions, and therefore interpretation of results, is difficult, especially when compounded by the presence of an intermediate colour due to the combination of two reactions. Because of this problem, normal mouse serum (NMS), BALB/c mouse serum and foetal calf serum (FCS) were tested for their effectiveness as blocking agents in a double-labelling procedure using two mouse anti-human MABs.

Are Ly-1 B cells important in autoimmune disease?

Although Ly-1 B cells produce autoantibodies and are found at elevated frequencies in certain autoimmune strains, very little is known about the role of these cells, if any, in autoimmune disease. In this publication, we summarize some recent findings relevant to Ly-1 B-cell development, clonal expansion and antibody production. We then consider the idea that Ly-1 B cells constitute the most primitive B-cell lineage in mammals and that the evolutionary niche occupied by these cells requires that they produce a basic set of autoantibodies that cross-react with common bacterial antigens.

Permanent alteration of the murine Ly-1 B repertoire due to selective depletion of Ly-1 B cells in neonatal animals.

Studies presented here demonstrate that paternal allotype Ly-1 B cells are permanently depleted following neonatal treatment with antibodies to the paternal IgM allotype. Paternal allotype conventional B cells, in contrast, are temporarily depleted by treatment with either anti-IgM or anti-IgD allotype antibodies and return rapidly to normal frequencies once the antibody treatment disappears. These differences are explained by basic developmental differences between Ly-1 B and conventional lineage B cells. That is, the conventional B cell population is replenished from Ig- precursors throughout life and, therefore, is only temporarily affected when depleted in neonates. The Ly-1 B cell population, in contrast, develops from Ig- progenitors during the prenatal and neonatal life but survives because it is exclusively self-replenishing in adults. Therefore, elimination of a population of Ly-1 B cells from neonates is tantamount to removing it forever. These findings suggest that while conventional B cells turn over rapidly and have an effectively unlimited repertoire, Ly-1 B cells express a repertoire whose composition is strongly influenced by neonatal conditions that favor or select against the retention of cells producing certain antibody molecules. Thus, Ly-1 B cells play a unique role in the immune system in that they retain indefinitely the history of the neonatal animal's immunological experience.

Feedback regulation of murine Ly-1 B cell development.

Studies presented here, conducted with allotype homozygotes, demonstrate the existence of a feedback mechanism that regulates development of Ly-1 B cells from immature progenitors. In the preceding study (P. A. Lalor et al., Eur. J. Immunol. 1989. 19:501), conducted with allotype heterozygotes, we showed that treating neonates with monoclonal antibody to the paternal allotype IgM depletes roughly half of the neonatal B cell population (i.e. those expressing the paternal IgM allotype) and that paternal allotype Ly-1 B cells specificically remain depleted for the life of the animal. Here we show that treating allotype homozygotes with the same antibody depletes all (rather than half) of the B cells and that, under these conditions, relatively normal numbers of Ly-1 B cells reappear shortly after the treatment antibody disappears. The recovery, we also show, is prevented by restoring allotype-congenic Ly-1 B cells to the treated homozygotes, i.e. by reconstituting treated neonates with allotype-congenic peritoneal cells, sorted Ly-1 B cells or a monoclonal population of Ly-1 B "tumor" cells. These findings in essence reveal a feedback mechanism through which mature Ly-1 B cells prevent further Ly-1 B cell development from Ig- precursors. This feedback regulation is independent of Ig secretion by the mature Ly-1 B cells, since the monoclonal Ly-1 B "tumor" population that prevents endogenous Ly-1 B development does not secrete Ig. Furthermore, it appears to be independent of Ly-1 B surface Ig specificity, since a monoclonal population is sufficient to block all Ly-1 B cell development. This mechanism appears to operate normally to fix the composition of the Ly-1 B population, which survives through self-replenishment in adults, in accord with conditions that influence Ly-1 B development during neonatal life.

Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity.

Long term B lineage chimeras are used here to study the origin of plasma cells in the mouse. Chimeric mice are constructed by reconstituting lethally irradiated mice with peritoneal cells (PerC) and bone marrow cells from congenic pairs of mice differing in Igh-C allotype. All conventional B cells in these mice express the allotype of the bone marrow donor and nearly all Ly-1 B lineage cells express the allotype of the PerC donor. FACS analysis and immunohistology of these mice shows that virtually all (sig+) B cells in peripheral lymphoid organs are derived from the bone marrow donor. However, despite this overwhelming number of bone marrow-derived B cells in these animals, immunohistological staining of lymphoid organs and gut shows that nearly half of the IgM, IgG, and IgA plasma cells derive from the PerC donor. These data demonstrate that the peritoneal cavity contains a major reservoir of self-replenishing cells that play a significant role in the mucosal immune response. The possibility that these are B cells that belong to the Ly-1 B lineage is discussed.