A novel method for quantitative measurements of gene expression in single living cells.

Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells.

Human peripheral blood CD4 T cell-engrafted non-obese diabetic-scid IL2rγ(null) H2-Ab1 (tm1Gru) Tg (human leucocyte antigen D-related 4) mice: a mouse model of human allogeneic graft-versus-host disease.

Graft-versus-host disease (GVHD) is a life-threatening complication of human allogeneic haematopoietic stem cell transplantation. Non-obese diabetic (NOD)-scid IL2rγ(null) (NSG) mice injected with human peripheral blood mononuclear cells (PBMC) engraft at high levels and develop a robust xenogeneic (xeno)-GVHD, which reproduces many aspects of the clinical disease. Here we show that enriched and purified human CD4 T cells engraft readily in NSG mice and mediate xeno-GVHD, although with slower kinetics compared to injection of whole PBMC. Moreover, purified human CD4 T cells engraft but do not induce a GVHD in NSG mice that lack murine MHC class II (NSG-H2-Ab1(tm1Gru), NSG-Ab°), demonstrating the importance of murine major histocompatibility complex (MHC) class II in the CD4-mediated xeno-response. Injection of purified human CD4 T cells from a DR4-negative donor into a newly developed NSG mouse strain that expresses human leucocyte antigen D-related 4 (HLA-DR4) but not murine class II (NSG-Ab° DR4) induces an allogeneic GVHD characterized by weight loss, fur loss, infiltration of human cells in skin, lung and liver and a high level of mortality. The ability of human CD4 T cells to mediate an allo-GVHD in NSG-Ab° DR4 mice suggests that this model will be useful to investigate acute allo-GVHD pathogenesis and to evaluate human specific therapies.

Skin allograft maintenance in a new synchimeric model system of tolerance.

Treatment of mice with a single donor-specific transfusion plus a brief course of anti-CD154 mAb uniformly induces donor-specific transplantation tolerance characterized by the deletion of alloreactive CD8+ T cells. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. To analyze the mechanisms underlying tolerance induction, maintenance, and failure in euthymic mice we created a new analytical system based on allo-TCR-transgenic hemopoietic chimeric graft recipients. Chimeras were CBA (H-2(k)) mice engrafted with small numbers of syngeneic TCR-transgenic KB5 bone marrow cells. These mice subsequently circulated a self-renewing trace population of anti-H-2(b)-alloreactive CD8+ T cells maturing in a normal microenvironment. With this system, we studied the maintenance of H-2(b) allografts in tolerized mice. We documented that alloreactive CD8+ T cells deleted during tolerance induction slowly returned toward pretreatment levels. Skin allograft rejection in this system occurred in the context of 1) increasing numbers of alloreactive CD8+ cells; 2) a decline in anti-CD154 mAb concentration to levels too low to inhibit costimulatory functions; and 3) activation of the alloreactive CD8+ T cells during graft rejection following deliberate depletion of regulatory CD4+ T cells. Rejection of healed-in allografts in tolerized mice appears to be a dynamic process dependent on the level of residual costimulation blockade, CD4+ regulatory cells, and activated alloreactive CD8+ thymic emigrants that have repopulated the periphery after tolerization.

Specific heat of Mg11B2: evidence for a second energy gap.

Measurements of the specific heat of Mg11B2 from 1 to 50 K, in magnetic fields to 9 T, give the Debye temperature, Theta = 1050 K, the coefficient of the normal-state electron contribution, gamma(n) = 2.6 mJ mol(-1) K-2, and a discontinuity in the zero-field specific heat of 133 mJ mol(-1) K-1 at T(c) = 38.7 K. The estimated value of the electron-phonon coupling parameter, lambda = 0.62, could account for the observed T(c) only if the important phonon frequencies are unusually high relative to Theta. At low T, there is a strongly field-dependent feature that suggests the existence of a second energy gap, about 4 times smaller than the major gap.

An unusual phase transition to a second liquid vortex phase in the superconductor YBa2Cu3O7.

A magnetic field penetrates a superconductor through an array of 'vortices', each of which carries one quantum of flux that is surrounded by a circulating supercurrent. In this vortex state, the resistivity is determined by the dynamical properties of the vortex 'matter'. For the high-temperature copper oxide superconductors (see ref.1 for a theoretical review), the vortex phase can be a 'solid', in which the vortices are pinned, but the solid can 'melt' into a 'liquid' phase, in which their mobility gives rise to a finite resistance. (This melting phenomenon is also believed to occur in conventional superconductors, but in an experimentally inaccessible part of the phase diagram.) For the case of YBa2Cu3O7, there are indications of the existence of a critical point, at which the character of the melting changes. But neither the thermodynamic nature of the melting, nor the phase diagram in the vicinity of the critical point, has been well established. Here we report measurements of specific heat and magnetization that determine the phase diagram in this material to 26 T, well above the critical point. Our results reveal the presence of a reversible second-order transition above the critical point. An unusual feature of this transition-namely, that the high-temperature phase is the less symmetric in the sense of the Landau theory-is in accord with theoretical predictions of a transition to a second vortex-liquid phase.

Viral infection abrogates CD8(+) T-cell deletion induced by costimulation blockade.

BACKGROUND: Treatment with a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (mAb) prolongs skin allograft survival in mice. It is known that prolongation of allograft survival by this method depends in part on deletion of alloreactive CD8(+) T cells at the time of tolerance induction. Recent data suggest that infection with lymphocytic choriomeningitis virus (LCMV) abrogates the ability of this protocol to prolong graft survival. METHODS: To study the mechanism by which viral infection abrogates allograft survival, we determined (1) the fate of tracer populations of alloreactive transgenic CD8(+) T cells and (2) the duration of skin allograft survival following treatment with DST and anti-CD154 mAb in the presence or absence of LCMV infection. RESULTS: We confirmed that treatment of uninfected mice with DST and anti-CD154 mAb leads to the deletion of alloreactive CD8(+) T cells and is associated with prolongation of skin allograft survival. In contrast, treatment with DST and anti-CD154 mAb in the presence of intercurrent LCMV infection was associated with the failure to delete alloreactive CD8(+) T cells and with the rapid rejection of skin allografts. The number of alloreactive CD8(+) cells actually increased significantly, and the cells acquired an activated phenotype. CONCLUSIONS: Interference with the deletion of alloreactive CD8(+) T cells mediated by DST and anti-CD154 mAb may in part be the mechanism by which viral infection abrogates transplantation tolerance induction.

Treatment of allograft recipients with donor-specific transfusion and anti-CD154 antibody leads to deletion of alloreactive CD8+ T cells and prolonged graft survival in a CTLA4-dependent manner.

A two-element protocol consisting of one donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb greatly prolongs the survival of murine islet, skin, and cardiac allografts. To study the mechanism of allograft survival, we determined the fate of tracer populations of alloreactive transgenic CD8+ T cells in a normal microenvironment. We observed that DST plus anti-CD154 mAb prolonged allograft survival and deleted alloreactive transgenic CD8+ T cells. Neither component alone did so. Skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus a depleting anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. We conclude that, in the presence of anti-CD154 mAb, DST leads to an allotolerant state, in part by deleting alloreactive CD8+ T cells. Consistent with this conclusion, blockade of CTLA4, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive transgenic CD8+ T cells. These results document for the first time that peripheral deletion of alloantigen-specific CD8+ T cells is an important mechanism through which allograft survival can be prolonged by costimulatory blockade. We propose a unifying mechanism to explain allograft prolongation by DST and blockade of costimulation.

NOD mice have a generalized defect in their response to transplantation tolerance induction.

A protocol consisting of a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (anti-CD40 ligand mAb) induces permanent islet allograft survival in chemically diabetic mice, but its efficacy in mice with autoimmune diabetes is unknown. Confirming a previous report, we first observed that treatment of young female NOD mice with anti-CD154 mAb reduced the frequency of diabetes through 1 year of age to 43%, compared with 73% in untreated controls. We also confirmed that spontaneously diabetic NOD mice transplanted with syngeneic (NOD-Prkdc(scid)/Prkdc(scid)) or allogeneic (BALB/c) islets rapidly reject their grafts. Graft survival was not prolonged, however, by pretreatment with either anti-CD154 mAb alone or anti-CD154 mAb plus DST. In addition, allograft rejection in NOD mice was not restricted to islet grafts. Anti-CD154 mAb plus DST treatment failed to prolong skin allograft survival in nondiabetic male NOD mice. The inability to induce transplantation tolerance in NOD (H2g7) mice was associated with non-major histocompatibility complex (MHC) genes. Treatment with DST and anti-CD154 mAb prolonged skin allograft survival in both C57BL/6 (H2b) and C57BL/6.NOD-H2g7 mice, but it was ineffective in NOD, NOD.SWR-H2q, and NOR (H2g7) mice. Mitogen-stimulated interleukin-1beta production by antigen-presenting cells was greater in strains susceptible to tolerance induction than in the strains resistant to tolerance induction. The results suggest the existence of a general defect in tolerance mechanisms in NOD mice. This genetic defect involves defective antigen-presenting cell maturation, leads to spontaneous autoimmune diabetes in the presence of the H2g7 MHC, and precludes the induction of transplantation tolerance irrespective of MHC haplotype. Promising islet transplantation methods based on overcoming the alloimmune response by interference with costimulation may require modification or amplification for use in the setting of autoimmune diabetes.

Prolonged survival of rat islet and skin xenografts in mice treated with donor splenocytes and anti-CD154 monoclonal antibody.

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and a brief course of anti-CD154 (anti-CD40 ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 days without further intervention. We now report adaptation of this protocol to the transplantation of islet and skin xenografts. We observed prolonged survival of rat islet xenografts in mice treated with donor-specific spleen cell transfusion and anti-CD154 monoclonal antibody (mAb). Challenge islet xenografts placed on these animals indicated that graft acceptance was species-specific but not strain specific. Spleen cells from recipients bearing intact grafts led to rejection of rat islet xenografts in scid mice, suggesting that graft acceptance was not due to complete clonal deletion of xenoreactive cells. We also observed prolonged survival of rat skin xenografts in mice treated with donor-specific transfusion and anti-CD154 mAb. Prolonged survival of skin xenografts was also species specific. We conclude that treatment with appropriately timed donor-specific transfusion and anti-CD154 mAb induces durable survival of both islet and skin xenografts in mice. Because this procedure is targeted directly at CD154, a co-activation molecule expressed predominantly by activated CD4+ T-cells, the results suggest that CD4+ cells have a major role in the cellular immune response to xenografts.

Long-term survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4(+) T cells, interferon-gamma, and CTLA4.

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and anti-CD154 (anti-CD40-ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 d without further intervention. The protocol induces long-term allograft survival, but the mechanism is unknown. We now report: (a) addition of thymectomy to the protocol permitted skin allografts to survive for > 100 d, suggesting that graft rejection in euthymic mice results from thymic export of alloreactive T cells. (b) Clonal deletion is not the mechanism of underlying long-term graft survival, as recipient thymectomized mice were immunocompetent and harbor alloreactive T cells. (c) Induction of skin allograft acceptance initially depended on the presence of IFN-gamma, CTLA4, and CD4(+) T cells. Addition of anti-CTLA4 or anti-IFN-gamma mAb to the protocol was associated with prompt graft rejection, whereas anti-IL-4 mAb had no effect. The role of IFN-gamma was confirmed using knockout mice. (d) Graft survival was associated with the absence of IFN-gamma in the graft. (e) Long-term graft maintenance required the continued presence of CD4(+) T cells. The results suggest that, with modification, our short-term protocol may yield a procedure for the induction of long-term graft survival without prolonged immunosuppression.

Prolonged survival of mouse skin allografts in recipients treated with donor splenocytes and antibody to CD40 ligand.

Combined treatment with antibody against CD40 ligand and one transfusion of donor splenocytes prolonged survival of fully mismatched BALB/c skin allografts on C57BL/6 recipients, with approximately 20% of grafts surviving > 100 days. In vitro alloresponsiveness in treated animals was reduced in the immediate post-transplantation period, but by day 100 was increased despite the presence of a successful allograft. The presence of alloreactivity on day 100 was confirmed in vivo by adoptive transfer, which suggests that our protocol had induced either a state of "split tolerance" or "graft accommodation." Mice with skin grafts that had survived for > or = 100 days revealed no evidence of lymphoid chimerism. Treatment with donor splenocytes and antibody against CD40 ligand permits long-term survival of highly antigenic donor skin allografts despite the presence of functionally intact alloreactive lymphocytes.

Modulation of ciliary beat frequency by neuropeptides from identified molluscan neurons.

Prior work in the nudibranch Tritonia diomedea indicated that certain identifiable pedal ganglion neurons (Pd5 and 6) innervating the foot synthesize three novel peptides (TPeps) that resemble Pedal peptide (Pep) identified in the sea hare Aplysia californica. We report here that when TPeps are applied directly to isolated ciliated patches of Tritonia diomedea foot epithelium, there is an increase in ciliary beating that normally drives locomotion. Exposure to TPeps also increases the ciliary beat frequency of cells isolated from the pedal epithelium, suggesting that the observed ciliomotor effects are direct and not mediated by intervening cells. Antibodies to TPep bind to specific cells of the brain and foot and to ciliated peripheral tissues in Tritonia diomedea and in the pulmonate gastropod Lymnaea stagnalis. We suggest, therefore, that TPeps may regulate the activity of ciliated cells responsible for pedal locomotion and other functions in gastropod molluscs.

Purification and sequencing of neuropeptides from identified neurons in the marine mollusc, Tritonia.

Neuropeptides were characterized in two similar identified neurons termed pedal 5 and 6 (Pd5 and Pd6). Both neurons appear white, a characteristic of peptidergic neurons, and send peripheral axons down several nerves that innervate the foot and control locomotion. Gel electrophoresis of neurons incubated with labeled amino acids indicated that individually dissected Pd5 and Pd6 neurons synthesized peptide precursors of the same size and processed them in parallel. Using HPLC, several absorbance peaks that had retention times typical of peptides were identified that were specific to extracts of Pd5 and Pd6. Three peptides were purified from extracts of many pooled Pd5 and Pd6 neurons. The complete sequences of two 15-amino acid peptides were obtained and the sequence of a third 15-amino acid peptide was inferred from the partial sequence of an apparent processing intermediate. Each of the three peptides show sequence homology to Aplysia pedal peptide (Pep). HPLC of neurons incubated with labeled amino acids demonstrated that Pd5 and Pd6 each synthesized all three sequenced peptides.

Induction of immunological tolerance to islet allografts.

T-cell dependent activation of resting B cells involves the interaction of gp39 on T cells with its receptor, CD40, on B cells. We administered either a combination of T-cell-depleted splenic lymphocytes and anti-gp39 monoclonal antibody or antibody alone to establish islet allografts in mice without continuous immunosuppression. Fully allogeneic H-2q FVB islets were permanently accepted by chemically diabetic H-2b C57BL/6 mice provided that the recipients were pretreated with both T-cell-depleted donor spleen cells and anti-gp39 antibody. Antibody alone was less effective in prolonging allograft survival, but we did observe that anti-gp39 mAb alone can exert an independent, primary effect on islet allograft survival that was dose dependent. Targeting gp39, in combination with lymphocyte transfusion, might prove suitable for tolerance induction and allotransplantation without immunosuppression.

Capture of Large Particles by Suspension-Feeding Scaleworm Larvae (Polychaeta: Polynoidae).

Most of the polychaete larvae in which feeding mechanisms have been studied feed using an opposed-band mechanism, capturing particles with prototrochal and metatrochal ciliary bands and transporting them to the mouth via a food groove. However, many other planktotrophic polychaete larvae lack a metatroch and food groove and thus must feed in a different way. In this latter group are the larvae of polynoid polychaetes, which not only lack a metatroch and food groove but also bear a bundle of long cilia (the oral brush) attached near the left side of the mouth. In feeding experiments with polystyrene beads and plankton, larvae of the polynoid Arctonoe vittata ingested larger particles (up to 60 {mu}m in diameter) than those ingested by the opposed-band feeding larvae of the serpulid Serpula vermicularis (up to 12 µm in diameter). Videotaped images of feeding A. vittata larvae showed that capture behavior was elicited as particles in a feeding current driven by the prototroch approached or contacted the larval episphere. Particles on or very near the episphere were disengaged by a recoiling motion of the larva and were then moved to the mouth, probably by the oral brush. This feeding mechanism may be widespread in the polychaete superfamily Aphroditacea, which includes about 10% of extant polychaete species.

Survival of mouse pancreatic islet allografts in recipients treated with allogeneic small lymphocytes and antibody to CD40 ligand.

Combined treatment with allogeneic small lymphocytes or T-depleted small lymphocytes plus a blocking antibody to CD40 ligand (CD40L) permitted indefinite pancreatic islet allograft survival in 37 of 40 recipients that differed from islet donors at major and minor histocompatibility loci. The effect of the allogeneic small lymphocytes was donor antigen-specific. Neither treatment alone was as effective as combined treatment, although anti-CD40L by itself allowed indefinite islet allograft survival in 40% of recipients. Our interpretation is that small lymphocytes expressing donor antigens in the absence of appropriate costimulatory signals are tolerogenic for alloreactive host cells. Anti-CD40L antibody may prevent host T cells from inducing costimulatory signals in donor lymphocytes or islet grafts.