Partial Maturation Research Articles

Skin irritants and contact sensitizers induce Langerhans cell migration and maturation at irritant concentration

Skin irritants and contact allergens reduce the number of Langerhans cells (LCs). It has been assumed that this reduction is due their migration to the draining lymph node (LN) for initiating immune sensitization in a host. Skin irritation, however, as opposed to contact allergy is not considered to be an immunological disease. Nevertheless, skin irritants are also known for their adjuvant-like effects on contact allergy, resulting in skin hypersensitivity reactions like toxic dermatitis. The human organotypic skin explant culture (hOSEC) model is used to study the characteristics of chemical-induced migration of CD1a(+) LCs out of the epidermis in relation to irritancy or toxicity. We analysed cells emigrating out of hOSEC for CD1a(+) LCs, CD83(+) mature dendritic cells (DCs) and CCR7(+) LN homing cells. After exposure to a toxic concentration of a non-immunogenic irritant, an increase of CD1a(+)CD83(+) LCs was found in the culture medium. A non-toxic concentration of an sensitizer induced an increase of CD1a(+) cells. About 50% of skin emigrating CD1a(+) LCs were CD83(-) (immature) but all were CCR7(+). Skin irritation by both non-allergenic and allergenic compounds induces LC migration and maturation. In contrast, only allergenic compounds induced LC migration with partial maturation at subtoxic concentration. This effectively demonstrates that irritation is physiologically needed stimuli for inducing LC maturation.

Hypochlorous acid enhances immunogenicity and uptake of allogeneic ovarian tumor cells by dendritic cells to cross-prime tumor-specific T cells.

Ovarian cancer commonly relapses after remission and new strategies to target microscopic residual diseases are required. One approach is to activate tumor-specific cytotoxic T cells with dendritic cells loaded with tumor cells. In order to enhance their immunogenicity, ovarian tumor cells (SK-OV-3, which express two well-characterized antigens HER-2/neu and MUC-1) were killed by oxidation with hypochlorous acid (HOCl). Treatment for 1 h with 60 microM HOCl was found to induce necrosis in all SK-OV-3 cells. Oxidized, but not live, SK-OV-3 was rapidly taken up by monocyte-derived dendritic cells, and induced partial dendritic cell maturation. Dendritic cells cultured from HLA-A2 healthy volunteers were loaded with oxidized SK-OV-3 (HLA-A2-) and co-cultured with autologous T cells. Responding T cells were tested for specificity after a further round of antigen stimulation. In ELISPOT assays, T cells produced interferon-gamma (IFN-gamma) in response to the immunizing cellular antigen, and also to peptides coding for MUC-1 and HER-2/neu HLA-A2 restricted epitopes, demonstrating efficient cross-presentation of cell-associated antigens. In contrast, no responses were seen after priming with heat-killed or HCl-killed SK-OV-3, indicating that HOCl oxidation and not cell death/necrosis per se enhanced the immunogenicity of SK-OV-3. Finally, T cells stimulated with oxidized SK-OV-3 showed no cross-reaction to oxidized melanoma cells, nor vice versa, demonstrating that the response was tumor-type specific. Immunization with oxidized ovarian tumor cell lines may represent an improved therapeutic strategy to stimulate a polyclonal anti-tumor cellular immune response and hence extend remission in ovarian cancer.

Partial maturation and robust ex vivo expansion of Umbilical Cord Blood (UCB) T cells: A step towards adoptive immunotherapy after UCB transplants

Partial maturation and robust ex vivo expansion of Umbilical Cord Blood (UCB) T cells: A step towards adoptive immunotherapy after UCB transplants

Aberrant maturation of mutant perforin underlies the clinical diversity of hemophagocytic lymphohistiocytosis.

Missense mutations in perforin, a critical effector of lymphocyte cytotoxicity, lead to a spectrum of diseases, from familial hemophagocytic lymphohistiocytosis to an increased risk of tumorigenesis. Understanding of the impact of mutations has been limited by an inability to express human perforin in vitro. We have shown, for the first time to our knowledge, that recombinant human perforin is expressed, processed appropriately, and functional in rat basophilic leukemia (RBL) cells following retroviral transduction. Subsequently, we have addressed how perforin missense mutations lead to absent perforin detection and impaired cytotoxicity by analyzing 21 missense mutations by flow cytometry, immunohistochemistry, and immunoblot. We identified perforin missense mutations with partial maturation (class 1), no apparent proteolytic maturation (class 2), and no recognizable forms of perforin (class 3). Class 1 mutations exhibit lytic function when expressed in RBL cells and are associated with residual protein detection and variable cytotoxic function in affected individuals, suggesting that carriers of class 1 alleles may exhibit more subtle immune defects. By contrast, class 3 mutations cause severely diminished perforin detection and cytotoxicity, while class 2 mutations have an intermediate phenotype. Thus, the pathologic mechanism of perforin missense mutation likely involves a protein dosage effect of the mature protein.

Ex Vivo Expansion of Umbilical Cord Blood T Cells for Adoptive Immunotherapy.

Background: Viral infections cause significant morbidity & mortality in patients undergoing unrelated allogeneic BMT transplantation before immune reconstitution is completed. It poses a greater risk for recipients of unrelated umbilical cord blood (uUCB) transplants as there is no established antiviral immunity in naïve UCB lymphocytes available for adoptive transfer. UCB T cells also lack Th1/Tc1 cytokines, Granzymes & Perforin which are prerequisites to control viral pathogens. Another major limitation of uUCBT is the lack of donor cells available for post-transplant donor leukocyte infusions (DLI) to boost immunity or induce GVL. However, a fraction of the uCB graft could be available for T cell expansion. In this study we evaluated the feasibility of ex vivo expansion of UCB T cells. We postulated that following expansion naïve T cells may mature & acquire a phenotype compatible with effector function as assessed by expression of essential cytokines & de novo expression of members of the granzyme-perforin pathway.Methods: Thawed UBC research samples with a leukocyte content <5% of an average UCB graft are processed. T cells are enriched with “EasySep” (StemCell Tech) to deplete CD14, CD16, CD19, CD56, & glycophorin A + cells. 5–7.5*105 T cells/ml are incubated with “CD3/28 T cell Expander” artificial APC beads (Dynal) in X Vivo-15 (BioWhittacker) + 200u/ml IL2 & 10% human serum in gas permeable bags. The initial purity of the T cells is 77–92%. The starting absolute T cell numbers ranged from 0.75 to 2*106 cells. Media & cytokines are added every other day to maintain a concentration of <2*106 cells/ml.Results: At the end of 14 days UCB T Cells expanded 67 fold +/− 36, n=6. There are significant alterations in phenotype over the 2 weeks (Table 1) with up to 40% of T cells in cell cycle. Compared to the starting resting UCB T cells the majority of expanded cells have acquired the phenotype of activated (HLA-DR+, CD25+ T cells) memory cells, at the expense of naive/recent thymic immigrants (CD45RA+/CD62+). There is an inverted CD4/CD8 ratio due to the higher expansion rate of CD8 T cells (p=0.0035) while there is no difference in apoptosis (p=0.57). However, they all retain expression of CD28 (96% ±8%) along with CD27. Although some T cells have acquired the capacity to secrete granzymes A and B these are still almost a log below normal adult peripheral blood (PB) values & perforin has not been detected. Similarly, while post expansion significantly more T cells secrete cytokines upon PMA + ionomycin stimulation (Table 1) they are below levels of adult PB.Conclusions: From our preliminary results we can demonstrate effective expansion & partial maturation of UCB T Cells. For example, if one starts with 2*106 total T cells & expands them 67 fold this could provide for DLI ~5*106 T cells/kg for an average pediatric patient (25kg). We are further optimizing & characterizing this model for T cell activity & repertoire. In sum, ex vivo expansion with CD3/CD28 co-stimulation may provide clinically relevant numbers T cells available for adoptive immunotherapy that have also undergone partial maturation.Characterization of Expanded T Cells as % of all LymphocytesVariableMedianSDCD3+99.80.1CD4+3511CD4+/CD8+2.32.8CD45RA+/RO−1311CD45RO+/RA−5522CD25+4221CD45RA+/CD62+3820CD45RA+/CD27+/CD8+5215CD45RA−/CD27+/CD8+4615KI67/CD8+429Ki67/CD4+327HLA DR+4013Granzyme A/CD8+5418Granzyme B/CD8+22Perforin/CD8+00

Differentiation of gonad maturation in sibling precocious males of the sea trout ( Salmo trutta m. trutta L.) in their first year of life

Age at maturity and dynamics of gonadal cycle vary among individuals in a standard population; the extent of that variability in a group of siblings is not known for any species. Therefore, the aim of the study was to find out how the differentiation of gonad development varies in a group of sibling fish. The study involved 176 seven-month-old sea trout parr (mean fork length of 11 cm) collected at a single time point during their first spawning season (late October) from the brook of Pniewa in Western Pomerania (Poland). Gonad maturation was determined from histological analysis which was interpreted according to specially developed criteria. The stage and forms of gonad maturation were diverse in the sibling group studied. Gonads of 52.8% males were not maturing (Class I), with only spermatogonia A present. The remaining males (47.2%) had gonads differing widely in the development, from beginning of maturation to completion of the reproductive cycle (Classes from II to VIII). In addition to the classes described, some males were observed to feature attempted maturation. It is difficult to provide an unequivocal estimate of the proportion of precocious males (mature at a young age, prior to the first descent to the sea) in the first year of life because of difficulties in categorizing males in Class II. It was predicted that 32.4% of the population would complete the cycle during the first spawning season. The body length and weight of the maturing males was significantly smaller ( P < 0.05) than that of the non-maturing males. Three forms of gonad maturation were identified. Gonads of a large group of precocious males (70%) developed incompletely (only some germ cell developed during the reproductive cycle), some of those gonads assumed the form of attempted maturation and partial maturation. Testes of the remaining 30% of the males underwent complete maturation. Gonads that undergo attempted maturation do not spermiate. It is only from microscope-aided analysis of the testes that the class of gonad development can be accurately evaluated and the extent of precocious maturation assessed, particularly in fish maturing for the first time. It is impossible to correctly identify maturing males of the underyearling sea trout on the basis of morphological examination of gonad appearance, abdominal massage to detect spermiation or gonadosomatic index value.

Tolerogenic Semimature Dendritic Cells Suppress Experimental Autoimmune Thyroiditis by Activation of Thyroglobulin-Specific CD4+CD25+ T Cells

Ex vivo treatment of bone marrow-derived dendritic cells (DCs) with TNF-alpha has been previously shown to induce partial maturation of DCs that are able to suppress autoimmunity. In this study, we demonstrate that i.v. administration of TNF-alpha-treated, semimature DCs pulsed with thyrogloblin (Tg), but not with OVA Ag, inhibits the subsequent development of Tg-induced experimental autoimmune thyroiditis (EAT) in CBA/J mice. This protocol activates CD4(+)CD25(+) T cells in vivo, which secrete IL-10 upon specific recognition of Tg in vitro and express regulatory T cell (Treg)-associated markers such as glucocorticoid-induced TNFR, CTLA-4, and Foxp3. These CD4(+)CD25(+) Treg cells suppressed the proliferation and cytokine release of Tg-specific, CD4(+)CD25(-) effector cells in vitro, in an IL-10-independent, cell contact-dependent manner. Prior adoptive transfer of the same CD4(+)CD25(+) Treg cells into CBA/J hosts suppressed Tg-induced EAT. These results demonstrate that the tolerogenic potential of Tg-pulsed, semimature DCs in EAT is likely to be mediated through the selective activation of Tg-specific CD4(+)CD25(+) Treg cells and provide new insights for the study of Ag-specific immunoregulation of autoimmune diseases.

Direct Effects of Polymyxin B on Human Dendritic Cells Maturation: THE ROLE OF IκB-α/NF-κB AND ERK1/2 PATHWAYS AND ADHESION

Polymyxin B is a lipopolysaccharide binding antibiotic used to inactivate potential lipopolysaccharide contaminations when evaluating the activity of different agents on innate immune cells. We report that polymyxin B is able to induce directly in monocyte-derived human dendritic cells (DCs) several functional and molecular modifications characteristic of DCs undergoing a maturation process. DCs incubated with polymyxin B up-regulate the expression of HLA class I and II, the co-stimulatory CD86 molecule, and show an increase in the fraction of adherent cells at short time, which persist at 48 h of incubation. Adhesion to the plate was required for the polymyxin B-induced DCs maturation. A transient activation of IkappaB-alpha/NF-kappaB and ERK1/2 pathways at short time and a further ERK1/2 activation at long term were also detected. Neither up-regulation of the maturation marker CD83 nor activation of p38 nor induction of cytokines secretion was observed in DCs treated with polymyxin B. We demonstrated that inhibition of IkappaB-alpha/NF-kappaB pathway abolishes polymyxin B effects. ERK1/2 inhibition instead allowed DCs treated with polymyxin B to progress in their maturation process as revealed by the increased up-regulation of the CD83 co-stimulatory molecules, the activation of p38, and the reduced adhesion to culture plates at 48 h of incubation. Our results indicate that polymyxin B induces a partial maturation of human DCs through increased adhesion to a substrate and activation of the IkappaB-alpha/NF-kappaB pathway. The increased ERK1/2 activation observed, even though correlating with the initial phases of the maturation process, actually inhibits the occurrence of full maturation.

Developmental Regulation of β-Carboline-Induced Inhibition of Glycine-Evoked Responses Depends on Glycine Receptor β Subunit Expression

In this work, we show that beta-carbolines, which are known negative allosteric modulators of GABA(A) receptors, inhibit glycine-induced currents of embryonic mouse spinal cord and hippocampal neurons. In both cell types, beta-carboline-induced inhibition of glycine receptor (GlyR)-mediated responses decreases with time in culture. Single-channel recordings show that the major conductance levels of GlyR unitary currents shifts from high levels (> or = 50 pS) in 2 to 3 days in vitro (DIV) neurons to low levels (<50 pS) in 11 to 14 DIV neurons, assessing the replacement of functional homomeric GlyR by heteromeric GlyR. In cultured spinal cord neurons, the disappearance of beta-carboline inhibition of glycine responses and high conductance levels is almost complete in mature neurons, whereas a weaker decrease in beta-carboline-evoked glycine response inhibition and high conductance level proportion is observed in hippocampal neurons. To confirm the hypothesis that the decreased sensitivity of GlyR to beta-carbolines depends on beta subunit expression, Chinese hamster ovary cells were permanently transfected either with GlyR alpha2 subunit alone or in combination with GlyR beta subunit. Single-channel recordings revealed that the major conductance levels shifted from high levels (> or = 50 pS) in GlyR-alpha2-transfected cells to low levels (<50 pS) in GlyR-alpha2+beta-containing cells. Consistently, both picrotoxin- and beta-carboline-induced inhibition of glycine-gated currents were significantly decreased in GlyR-alpha2+beta-transfected cells compared with GlyR-alpha2-containing cells. In summary, we demonstrate that the incorporation of beta subunits in GlyRs confers resistance not only to picrotoxin but also to beta-carboline-induced inhibition. Furthermore, we also provide evidence that hippocampal neurons undergo in vitro a partial maturation process of their GlyR-mediated responses.

Maturation of dendritic cells in the presence of living, apoptotic and necrotic tumour cells derived from squamous cell carcinoma of head and neck

Dendritic cells (DC) have been recently used as vaccines for stimulation of tumour-specific immunity in various types of cancer. Since data about interactions of DC with tumour cells derived from head and neck cancer are not available, in our study we investigated the effects of head and neck squamous cell carcinoma (HNSCC) cell lines on the maturation of DC. We found that immature DC efficiently internalise necrotic cells, but not living and apoptotic tumour cells. Although apoptotic cells induced a partial maturation of DC, they were not able to stimulate the secretion of IL-12. In contrast, necrotic tumour cell preparations from all three HNSCC cell lines induced the mature phenotype and IL-12 production by DC. Moreover, necrotic cells synergistically augmented stimulatory effects of monocyte-conditioned medium on the maturation of DC. Thus, DC-based vaccination utilizing necrotic tumour cells as a source of tumour antigens, even in combination with inflammatory stimulus, seems to be a suitable strategy for adjuvant immunotherapy in HNSCC.

Recombinant adenovirus-transduced human dendritic cells engineered to secrete interleukin-10 (IL-10) suppress Th1-type responses while selectively activating IL-10—producing CD4 + T cells

Recombinant adenoviruses (rAd) are efficient tools for genetic modification of human dendritic cells (DC) in vitro. Infection of DCs by rAd encoding β-galactosidase (βgal) results in partial maturation of DCs, as witnessed by the upregulation of major histocompatibility complex and costimulatory molecules. Accordingly, these DCs are more potent stimulators of Th1-type proliferative responses. We now demonstrate that infection of immature DCs with rAd encoding human interleukin (IL)-10 results in the secretion by the DCs of large amounts of IL-10, while not affecting expression of activation markers indicative of partial DC maturation. In contrast to rAd-βgal–infected DCs, rAdIL-10–infected DCs are very poor stimulators of monoclonal and polyclonal Th1-type responses. Instead, stimulation of nonpolarized CD4 + T-cell cultures with rAdIL-10–infected DCs selectively activates and expands an IL-10–producing CD4 + T-cell subset capable of suppressing Th1 responses in vitro. Our data argue that rAd-infected human DCs genetically engineered to produce IL-10 may be exploited for the modulation of harmful Th1-type responses in transplantation and autoimmune diseases.

Spindle pole body duplication in fission yeast occurs at the G1/S boundary but maturation is blocked until exit from S by an event downstream of cdc10+.

The regulation and timing of spindle pole body (SPB) duplication and maturation in fission yeast was examined by transmission electron microscopy. When cells are arrested at G1 by nitrogen starvation, the SPB is unduplicated. On release from G1, the SPBs were duplicated after 1-2 h. In cells arrested at S by hydroxyurea, SPBs are duplicated but not mature. In G1 arrest/release experiments with cdc2.33 cells at the restrictive temperature, SPBs remained single, whereas in cells at the permissive temperature, SPBs were duplicated. In cdc10 mutant cells, the SPBs seem not only to be duplicated but also to undergo partial maturation, including invagination of the nuclear envelope underneath the SPB. There may be an S-phase-specific inhibitor of SPB maturation whose expression is under control of cdc10(+). This model was examined by induction of overreplication of the genome by overexpression of rum1p or cdc18p. In cdc18p-overexpressing cells, the SPBs are duplicated but not mature, suggesting that cdc18p is one component of this feedback mechanism. In contrast, cells overexpressing rum1p have large, deformed SPBs accompanied by other features of maturation and duplication. We propose a feedback mechanism for maturation of the SPB that is coupled with exit from S to trigger morphological changes.

Proportional oocyte nuclear maturation in relation to ICSI outcome

Depending on different stimulation protocols and varying patient response, about 70–80% of the oocytes are metaphase II at egg retrieval. When the contribution of the immature oocytes is higher, particularly when over 50%, this is cause of great distress to physicians and patients. In standard in vitro fertilization, the proportion of mature oocytes can be determined only on the day of fertilization assessment, whereas in the case of ICSI this becomes obvious immediately after cumulus removal, allowing insemination of only mature oocytes. In the present study, we aimed to assess the impact of a high proportion of immature oocytes present at retrieval on ICSI outcome. Patients who underwent the ICSI procedure were classified retrospectively according to the proportion of immature oocytes at the time of retrieval. Term outcomes were fertilization, implantation, and pregnancies. Couples treated by ICSI from September 1993 to December 2001 were included in this study. In order to exclude confounding factors related to maternal age or severe male factor, only women ≤35 years of age and only ejaculated spermatozoa were used. Ovarian stimulation was performed with a GnRH-agonist/antagonist and gonadotropins, with oocytes retrieved approximately 36 hours after hCG administration. ICSI was then performed only on MII oocytes in a standard fashion. Fertilization was assessed 16–18 hours after insemination and good quality embryos were transferred to the patients either on day 3 or day 5. Patients were stratified into three groups: with 100% maturation (complete), those with 50% of the oocytes matured (partial), and those with only 30% mature (minimal). A total of 2,368 ICSI cycles using ejaculated spermatozoa were assessed, with a similar maternal age in all groups and an overall average maternal age of 32.1 ± 3 yrs (range 18–35 yrs). The number of cycles for the complete maturation group was 388 (16.4%), for the partial maturation was 116 (4.9%), and for the minimal maturation was 31 (1.3%). The total number of oocytes retrieved was 3,274, 624, and 87, respectively, while the average number of oocytes injected was 8.4, 5.5, and 3.9. The fertilization rates and patterns were not different among the three cohorts, being 73.5, 69.7, and 76.9%, respectively, and the implantation rate was directly related to number of mature oocytes (28.9, 31.8, and 23.8%). Proportional differences were found in the pregnancy (46.4, 50.0, and 29.0%; P < 0.01) and in delivery rates (44.3, 44.8, and 25.8%; P < 0.01). Thus, while the outcomes for the complete and partial maturation gorups were comparable, the minimal maturation group had a lower rate of success. Within the cohort of oocytes retrieved the proportion of those at the MII stage did not affect fertilization outcome as long as at least half of them were mature. Only when ∼33% of mature oocytes was present was when the ICSI outcome compromised. Although the lower number of mature oocytes did not impair quality of any embryos eventually produced as judged by the rates of implantation, this still impairs pregnancy outcome because of a lower number of embryos transferred.

A DAP12-mediated pathway regulates expression of CC chemokine receptor 7 and maturation of human dendritic cells.

Gene targeting of the adaptor molecule DAP12 in mice caused abnormal distribution and impaired antigen presentation capacity of dendritic cells (DCs). However, the DAP12-associated receptors expressed on DCs and their functions have not been identified yet. Here we show that the triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor on human monocyte-derived DCs, which is associated with DAP12. TREM-2/DAP12 promotes upregulation of CC chemokine receptor 7, partial DC maturation, and DC survival through activation of protein tyrosine kinases and extracellular signal-regulated kinase. In contrast to Toll-like receptor-mediated signaling, TREM2/DAP12 stimulation is independent of nuclear factor-kappaB and p38 stress-activated protein kinase. This novel DC activation pathway may regulate DC homeostasis and amplify DC responses to pathogens, explaining the phenotype observed in DAP12-deficient mice.

The TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity.

LIGHT is a recently identified member of the TNF superfamily that is up-regulated upon activation of T cells. Herpesvirus entry mediator, one of its receptors, is constitutively expressed on immature dendritic cells (DCs). In this report, we demonstrate that LIGHT induces partial DC maturation as demonstrated by Ag presentation and up-regulation of adhesion and costimulatory molecules. LIGHT-stimulated DCs show reduced macropinocytosis and enhanced allogeneic stimulatory capacity but fail to produce significant amounts of IL-12, IL-6, IL-1beta, or TNF-alpha compared with unstimulated DCs. However, LIGHT cooperates with CD154 (CD40 ligand) in DC maturation, with particular potentiation of allogeneic T cell proliferation and cytokine secretion of IL-12, IL-6, and TNF-alpha. Moreover, LIGHT costimulation allows DCs to prime in vitro-enhanced specific CTL responses. Our results suggest that LIGHT plays an important role in DC-mediated immune responses by regulating CD154 signals and represents a potential tool for DC-based cancer immunotherapy.

Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-κB pathway

Dendritic cells (DC) are key components of innate and adaptive immune responses. The identity of endogenous signals that activate DC is a crucial and unresolved question. We report here that heat shock proteins (HSP), the most abundant and conserved mammalian molecules, constitute such an internal signal. Necrotic but not apoptotic cell death leads to release of HSP gp96, calreticulin, hsp90 and hsp70. HSP stimulate macrophages to secrete cytokines, and induce expression of antigen-presenting and co-stimulatory molecules on the DC. The HSP gp96 and hsp70 act differentially, and each induces some but not all molecules. HSP interact with these antigen-presenting cells through the highly conserved NF-kappa B pathway. As HSP are intracellular, abundant and soluble, their presence in the extra-cellular milieu and the consequent activation of antigen-presenting cells (APC) constitutes an excellent mechanism for response to cell death. As HSP are conserved from bacteria to mammals, the ability of HSP to activate APC provides a unified mechanism for response to internal and external stimuli.

The influence of mechanical stimulus on the pattern of tissue differentiation in a long bone fracture — an FEM study

2D, coronal plane, finite elements models (FEMs) were developed from orthogonal radiographs of a diaphyseal tibial fracture and its reparative tissue at four different time points during healing. Each callus was separated into regions of common tissue histology by computerised radiographic analysis. Starting point values of tissue material properties from the literature were refined by the model to simulate exactly the mechanical behaviour of the subject's callus and bone during loading. This was achieved by matching measured inter-fragmentary displacements with calculated inter-fragmentary forces. Stress and strain distributions in the callus and bone were calculated from peak inter-fragmentary displacements measured during natural walking activity, and were correlated with the subsequently observed pattern of tissue differentiation and maturation of the callus. The growth and stiffening of the external callus progressively reduced the inter-fragmentary gap strain. Partial maturation of the gap tissue was apparent only one week before fixator removal. Principal stresses in the callus were compared with `yield stresses' in corresponding tissue from the literature. This indicated the presence of stress concentrations medial and lateral to the fracture-gap, which probably caused tissue damage during normal activity levels. Tissue damage may also have precipitated partial structural failure of the callus, both of which were believed to have delayed healing during the middle third of the fixation period. Had the fixation device provided greater inter-fragmentary support during early healing, this may have prevented callus failure and the consequent delay in healing. A further benefit of this would have been the reduction of the initially high intra-gap tissue strains to a magnitude more conducive to earlier maturation of the bridging tissue that united the bone.

Familial Acute Myeloid Leukemia with Monosomy 7: Late Onset and Involvement of a Multipotential Progenitor Cell

Familial acute myeloid leukemia (AML) with monosomy 7 is a rare syndrome with fewer than 10 families reported. The salient features included a young median age (8 years) at presentation, equal sex preference, and occurrence of cytopenias and myelodysplasia in nonleukemic family members. Owing to its rarity and the fact that many cases were reported quite some time ago, detailed clinicopathologic features of familial monosomy 7 were not available. We describe a family with three siblings affected by AML in whom monosomy 7 was demonstrated. This family showed several unique features, including the late onset of AML (at 34 and 37 years of age in two siblings), and the presence of an antecedent myelodysplastic phase before leukemia development. With fluorescence in situ hybridization, the monosomy 7 clone was shown to be capable of partial maturation, which was consistent with the biologic behavior of myelodysplasia. These observations suggest that familial leukemia with monosomy 7 is probably a multistep leukemogenic process in which monosomy 7 might be but one of the critical steps. Finally, the prognosis in these cases was poor, suggesting that more aggressive therapy may be needed to improve treatment outcome.

Evidence for incomplete replication of a penguin poxvirus in cells of mammalian origin.

The recent discovery of a novel poxvirus [penguin-pox virus (PPV)] from Jackass penguins offers the potential of a unique candidate vaccine vector for use in mammals. Infectivity studies were therefore undertaken using a number of mammalian cell lines and chick embryo fibroblasts (CEF). It was shown that the simian CV-1 cell line was able to support replication of the PPV DNA, but no infectious progeny virus could be recovered from the infected cells. Electron microscopy was used to establish the extent of virus morphogenesis in CV-1 cells as compared to that in both chorio-allantoic membranes (CAMs) of hens' eggs and CEF cells. It appears that CV-1 cells are able to support partial maturation of PPV, but that morphogenesis does not proceed to the stage of mature infectious particles. Vaccinia virus promoters were successful in achieving transient gene expression in PPV-infected cells.

BCL-XL-regulated apoptosis in T cell development.

Thymocyte differentiation progresses through well-defined stages in which apoptosis is central to the selection of a functional TCR repertoire. In the present study, we explored the developmental effects of BCL-XL, a repressor of apoptosis. We found that endogenous BCL-XL is down-regulated by both positive and negative selection signals at the CD4+ CD8+ stage of thymocyte development. We examined the role of BCL-XL regulatable apoptosis in T cell development in the context of an alpha beta TCR transgene, Rag-1 deficiency and the scid model. We found that BCL-XL expression promoted accumulation of CD8 single-positive thymocytes even in MHC class II-restricted TCR transgenic mice. However, the apoptotic resistance conferred by BCL-XL could not fully substitute for TCR-mediated positive selection signals nor did it prevent negative selection. Overexpression of BCL-XL promoted partial maturation of CD4-CD8- thymocytes to CD4+CD8+ cells in a Rag-deficient, but not a scid background. Thus, TCR-mediated signals mediate the regulation of endogenous BCL-XL during thymocyte development, indicating that the principal protection of double-positive thymocytes by BCL-XL occurs prior to selection. The impact of BCL-XL on the CD8 but not CD4 lineage supports the asymmetric model of lineage commitment. Moreover, several critical control points in T cell development could be distinguished as BCL-XL responsive or unresponsive.