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The crisis caused by COVID-19 has profoundly affected human activities around the globe, and the Galapagos Islands are no exception. The impacts on this archipelago include the impairment of tourism and the loss of linkages with the Ecuadorian mainland, which has greatly impacted the local economy. The collapse of the local economy jeopardized livelihoods and food security, given that many impacts affected the food supply chain. During the crisis, the artisanal fishers of the Galapagos showed a high capacity to adapt to the diminishing demand for fish caused by the drastic drop in tourism. We observed that fishers developed strategies and initiatives by shifting roles, from being mainly tourism-oriented providers to becoming local-household food suppliers. This new role of fishers has triggered an important shift in the perception of fishers and fisheries in Galapagos by the local community. The community shifted from perceiving fisheries as a sector opposed to conservation and in conflict with the tourism sector to perceiving fisheries as the protagonist sector, which was securing fresh, high-quality protein for the human community. This study explores the socio-economic impacts and adaptations of COVID-19 on Galapagos' artisanal fisheries based on a mixed methods approach, including the analysis of fisheries datasets, interviews, surveys, and participant observation conducted during and after the lockdown. We illustrate the adaptive mechanisms developed by the sector and explore the changes, including societal perceptions regarding small-scale fisheries in the Galapagos. The research proposes strategies to enhance the Galapagos' economic recovery based on behaviors and traits shown by fishers which are considered potential assets to build-up resilience.The online version contains supplementary material available at 10.1007/s40152-022-00268-z.

Mangroves provide many benefits to human welfare, but they are disappearing rapidly; Ecuador and countries within the Tropical Eastern Pacific (TEP) region have lost over 40% of their mangrove coverage in the last 40 years. One reason for this destruction is that the benefits of mangroves have not been valued in a way that policymakers and markets can understand. Here, we present the first economic valuation of multiple ecosystem services (ES) for Ecuador and the TEP using the Galapagos mangroves as a case study. We focused on three ES of high value and policy relevance: carbon storage, support for small-scale fisheries, and mangrove-based tourism. Our data suggest that over 778,000 tons of carbon are stored in Galapagos mangroves, with mean belowground carbon being 211.03 ± 179.65 Mg C/ha, valued at $2940/ha or $22,838/ha depending on the valuation methodology. We identified mangrove-dependent fish targeted by the local finfish fishery, with net benefits of $245 ha−1, making this fishery the second most profitable in the Archipelago. The value of mangrove-based recreation was estimated at $16,958/ha, contributing $62 million to the industry. By accounting for stakeholders and existing property rights, our results allow for the discussion of institutionalizing ES payments for the Galapagos Islands.

In spite of several policies to promote fisheries management in the Galapagos Marine Reserve (GMR), in the past there has been a collapse or near collapse of the two most important fisheries: the sea cucumber and the lobster fishery respectively. Currently, there is particular concern for the Galapagos Sailfin Grouper (Mycteroperca olfax), a regionally endemic species showing signs of severe decline and targeted by the white-fin fishery, a multi-species coastal fishery of high market value. Within this context, the Galapagos National Park Directorate (GNPD) and local NGOs have designed a policy consistent in a product certification scheme aiming to shift fishing efforts from depleted coastal fisheries to healthier pelagic stocks, whilst supporting artisanal fisher’s livelihoods. Using a discrete choice experiment and accompanying survey data, the goal of this research is to analyze the preferences and tradeoffs made by individuals across attributes when purchasing certified fish in the Galapagos. In particular, we account for pro-environmental attitudes within a WTP space model specification and estimate the expected price premiums that tourists would be willing to pay for yellowfin tuna in the area in light of specific characteristics of its local value chain. We find that tourists visiting the Galapagos hold pro environmental attitudes as measured by the New-ecological paradigm and that there is a considerable increase in consumer’s utility from a certification scheme. Tourists care about the level of bycatch associated with their consumption, food safety, quality, and whether is locally sourced, and would be willing to pay for these attributes. This evidence, driven by location specific characteristics of consumers and tourists, supports the design of market-based mechanisms for sustainable yellow fin tuna fisheries in the Galapagos

Background. Fms-related tyrosine kinase 3 (Flt3)-ligand (FL) promotes the proliferation, differentiation, development, and mobilization ofhematopoietic cells. We previously found that FL-mobilized hematopoietic stem cells (HSC) engraft efficiently, whereas FL-expanded bone marrow HSC do not. The function of FL-mobilized c-Kit + Sca-1 + Lin - (KSL) subpopulations has not been systematically evaluated. A precise definition of the repopulating ability is needed to define which HSC subpopulations are critical for long-term chimerism and tolerance induction. FL significantly mobilized c-Kit hi and c-Kit lo Sca-1 + Lin - cells into peripheral blood (PB). Here, we evaluated the influence of Flt3 expression on long-term repopulating ability of HSC subpopulations. Methods. c-Kit hi or c-Kit lo KSL cells were sorted from PB of FL-treated green fluorescent protein-positive donors. The function of these cells was evaluated using competitive reconstitution assays, colony-forming units spleen, and colony forming cell assays. The function of c-Kit hi CD34 - Flt3 - KSL, c-Kit hi CD34 + Flt3 - KSL, c-Kit hi CD34 + Flt3 + KSL were investigated in an in vivo transplantation model. Results. Only FL-mobilized PB c-Kit hi KSL cells exhibited high spleen colony-forming unit activity, generated high numbers of both lymphoid and myeloid colonies in vitro, and rescued ablated recipients. FL-mobilization expanded both c-Kit hi CD34 + Flt3 - cells (short-term HSC) and c-Kit hi CD34-Flt3- KSL cells (long-term HSC). There was a significant decrease in c-Kit hi CD34 + Flt3 + KSL late multipotent progenitors in PB. A combination of c-Kit hi CD34 + Flt3 - and c-Kit hi CD34-Flt3- KSL cells offered the most effective rescue of ablated recipients. Conclusions. These data suggest that engraftment of purified HSC is influenced by both short- and long-term repopulating populations and that Flt3 expression may be useful for selecting the most critical HSC subpopulations for transplantation.

The use of tolerogenic cells as an approach to induce tolerance to solid organ allografts is being aggressively pursued. A major limitation to the clinical application of cell-based therapies has been the ability to obtain sufficient numbers and also preserve their tolerogenic state. We previously reported that small numbers of bone marrow-derived CD8+/TCR− graft facilitating cells (FC) significantly enhance hemopoietic stem cell (HSC) engraftment in allogeneic and syngeneic recipients. Although the majority of FC resemble precursor plasmacytoid dendritic cells (p-preDC), p-preDC do not replace FC in facilitating function. In the present studies, we investigated the mechanism of FC function. We show for the first time that FC significantly enhance HSC clonogenicity, increase the proportion of multipotent progenitors, and prevent apoptosis of HSC. These effects require direct cell:cell contact between FC and HSC. Separation of FC from HSC by transwell membranes completely abrogates the FC effect on HSC. p-preDC FC do not replace FC total in these effects on HSC function. FC produce TNF-α, and FC from TNF-α-deficient mice exhibit impaired facilitation in vivo and loss of the in vitro effects on HSC. Neutralizing TNF-α in FC similarly blocks the FC effect. The antiapoptotic effect of FC is associated with up-regulation of Bcl-3 transcripts in HSC and blocking of TNF-α is associated with abrogation of up-regulation of Bcl-3 transcripts. These data demonstrate a critical role for TNF-α in mediating FC function. FC may have a significant impact upon the safe use of chimerism to establish tolerance to transplanted organs and tissue.

Facilitating cells (CD8+/TCR−) (FCs) enhance engraftment of limiting numbers of hematopoietic stem cells (HSCs). The primary component of FCs is precursor-plasmacytoid dendritic cells (p-preDCs), a tolerogenic cell expanded by Flt3-ligand (FL). In this study, we evaluated the function and composition of FL-expanded FCs. FL treatment resulted in a significant increase of FCs in bone marrow (BM) and peripheral blood (PB). When FL-expanded FCs were transplanted with c-Kit+/Sca-1+/Lin− (KSL) cells into allogeneic recipients, BM-FCs exhibited significantly impaired function whereas PB-FCs were potently functional. A significant upregulation of P-selectin expression and downregulation of VCAM-1 (vascular cell adhesion molecule 1) were present on FL-expanded PB-FCs compared with FL BM-FCs. Stromal cell–derived factor-1 (SDF-1), and CXCR4 transcripts were significantly increased in FL PB-FCs and decreased in FL BM-FCs. Supernatant from FL PB-FCs primed HSC migration to SDF-1, confirming production of the protein product. The FL PB-FCs contained a predominance of p-preDCs and natural killer (NK)–FCs, and NK-FCs were lacking in FL BM-FCs. The impaired function for BM-FCs was restored within 5 days after cessation of treatment. Taken together, these data suggest that FCs may enhance HSC homing and migration via the SDF-1/CXCR4 axis and adhesion molecule modulation. These findings may have implications in development of strategies for retaining function of ex vivo manipulated FCs and HSCs.

Facilitating cells (CD8+/TCR-) (FCs) enhance engraftment of limiting numbers of hematopoietic stem cells (HSCs). The primary component of FCs is precursor-plasmacytoid dendritic cells (p-preDCs), a tolerogenic cell expanded by Flt3-ligand (FL). In this study, we evaluated the function and composition of FL-expanded FCs. FL treatment resulted in a significant increase of FCs in bone marrow (BM) and peripheral blood (PB). When FL-expanded FCs were transplanted with c-Kit+/Sca-1+/Lin- (KSL) cells into allogeneic recipients, BM-FCs exhibited significantly impaired function whereas PB-FCs were potently functional. A significant upregulation of P-selectin expression and downregulation of VCAM-1 (vascular cell adhesion molecule 1) were present on FL-expanded PB-FCs compared with FL BM-FCs. Stromal cell-derived factor-1 (SDF-1), and CXCR4 transcripts were significantly increased in FL PB-FCs and decreased in FL BM-FCs. Supernatant from FL PB-FCs primed HSC migration to SDF-1, confirming production of the protein product. The FL PB-FCs contained a predominance of p-preDCs and natural killer (NK)-FCs, and NK-FCs were lacking in FL BM-FCs. The impaired function for BM-FCs was restored within 5 days after cessation of treatment. Taken together, these data suggest that FCs may enhance HSC homing and migration via the SDF-1/CXCR4 axis and adhesion molecule modulation. These findings may have implications in development of strategies for retaining function of ex vivo manipulated FCs and HSCs.

Background In the present study we examined the effect of the timing of marrow infusion on engraftment in nonmyeloablatively conditioned mice. Methods B10 mice were conditioned with decreasing doses of total body irradiation (TBI) and reconstituted with bone marrow cells (BMCs) from major histocompatibility complex-disparate donor B10.BR mice at 0 or 6 hr, or on days 1, 2, 3, 4, 5, 8, and 12 with respect to TBI. Results After undergoing conditioning with 700 cGy TBI and transplantation with 15 x 10(6) BMCs, 100% of recipients engrafted if the marrow was infused between 0 and 4 days after TBI. For lower doses of TBI, a delay in infusion of the marrow after TBI conditioning was associated with a significant increase in engraftment. Significantly less engraftment was achieved in animals conditioned with 600 cGy TBI if the marrow was infused at 0 or 6 hr compared with a 1- to 4-day delay. When the TBI was decreased to 500 cGy, engraftment occurred only when the transplant was performed between days 2 and 8. The highest proportion of recipients engrafted when the marrow was infused on day 4. This enhanced engraftment after a delay in marrow infusion is associated with a significant reduction in host mixed lymphocyte reaction reactivity and is correlated inversely with serum levels of interleukin-6 in the recipient. Conclusions These data demonstrate for the first time that a delay between conditioning and marrow infusion significantly improves allogeneic engraftment in nonmyeloablatively conditioned recipients and reduces the total conditioning required.

Conjugation of dextran-coated superparamagnetic iron oxide (SPIO) particles with transactivator protein (Tat)-peptide and fluorescein isothiocyanate (FITC) allows cells to readily uptake SPIO particles. This makes possible high-resolution, real-time imaging of these cells by magnetic resonance imaging (MRI). First, we need to understand how various subpopulations take up and maintain SPIO particles. In this report, we have focused on differences in T cells, B cells, and macrophages with respect to cross-linked (CL)-SPIO Tat-FITC particle uptake over 72 hours. We have found that cells quickly take up the particles and that the bead loss that does occur is not related to cell death or apoptosis. In contrast with reports in the literature, we have observed migration of the Tat-peptide conjugates primarily to the cytoplasm rather than the nucleus.

We describe techniques to detect nuclear transcription factors in thymocyte subsets using flow cytometry. We have adapted a method that minimizes autofluorescence of fixed cells, thereby allowing the detection of proteins expressed at low levels. An accompanying method in which the cytoplasm is removed from stained cells allows the confirmation of nuclear localization. These methods combine to provide a sensitive alternative approach for detecting nuclear proteins within heterogeneous cell populations.

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of alphabeta-TCR(+), gammadelta-TCR(+), alphabeta- plus gammadelta-TCR(+), CD8(+), or CD4(+) cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4(+) T cells. Mice lacking both alphabeta- and gammadelta-TCR(+) cells engrafted without conditioning, suggesting that both alphabeta- and gammadelta-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8(-) cell was targeted by the CyP conditioning. The CD8(+) cell effector function is mechanistically different from that for conventional T cells, and independent of CD4(+) T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.

The broad objective of the research presented here is to develop a noncatalytic plasmid maintenance system for the stabilization of multicopy expression plasmids encoding foreign antigens in a Salmonella typhi live-vector vaccine strain such as CVD 908- htrA . We have enhanced the maintenance of expression plasmids at two independent levels. First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes expressed from multicopy plasmids; we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the noncatalytic hok-sok plasmid addiction system from the antibiotic resistance factor pR1. We also included at least one naturally occurring plasmid partition function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to accomplish this, we incorporated either the par locus from pSC101, the parA locus from pR1, or both. We monitored the stability of optimized expression plasmids within CVD 908- htrA by quantitating expression of a variant of green fluorescent protein (GFPuv) by using flow cytometry. In this report, we demonstrate the utility of this novel plasmid maintenance system in enhancing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthesis also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are discussed.

The cytokine production patterns of human peripheral blood mononuclear cells (PBMC) in response to Salmonella typhi flagella (STF) were examined in culture supernatants of PBMC stimulated with STF. Consistent with previous findings in volunteers vaccinated with aroC aroD deletion mutants of S. typhi , PBMC from volunteers immunized with the licensed live Ty21a S. typhi vaccine secreted gamma interferon following exposure to STF. Stimulation with STF induced rapid de novo synthesis of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), followed by IL-6 and IL-10. Trypsin treatment of STF abrogated their effects, while polymyxin B had no effect. Intracellular cytokine measurements of STF-stimulated PBMC revealed the existence of monocyte subpopulations that produce only TNF-α, IL-1β or both cytokines. Moreover, STF markedly decreased the percentage of CD14 + cells. These data demonstrate that STF are powerful monocyte activators which may have important implications for vaccine development and for understanding the pathogenesis of S. typhi infection.

Vibrio vulnificus is a human pathogen whose virulence has been associated with the expression of capsular polysaccharide (CPS). Multiple CPS types have been described; however, virulence does not appear to correlate with a particular CPS composition. Reversible-phase variation for opaque and translucent colony morphologies is characterized by changes in CPS expression, as suggested by electron microscopy of cells stained nonspecifically with ruthenium red. Isolates with opaque colony morphologies are virulent and appear to be more thickly encapsulated than naturally occurring translucent-phase variants, which have reduced, patchy, or absent CPS. Previously, we have shown that the virulence of translucent-phase variants was intermediate between opaque-phase variants and acapsular transposon mutants, suggesting a correlation between virulence and the amount of CPS expressed. In the present study, CPS expression of phase variants and genetically defined mutants of V. vulnificus M06-24/O was examined by using a CPS-specific monoclonal antibody with an enzyme-linked immunosorbent assay, flow cytometry, and immunoelectron microscopy. Semiquantitative analyses of CPS expression correlated well among these assays, confirming that the translucent-phase variant was intermediate in CPS expression and retained type I CPS-specific epitopes. Cell surface expression of CPS varied with the growth phase, increasing during logarithmic growth and declining in stationary culture. Significantly greater CPS expression ( P = 0.026) was observed for cells grown at 30°C than for those at 37°C. These studies confirm that phase variation and virulence in V. vulnificus correlate with the amount of CPS expressed and demonstrate the fluidity of bacterial polysaccharide expression in response to environmental conditions.

A key function of monocytes/macrophages (Mφ) is to present antigens to T cells. However, upon interaction with bacteria, Mφ lose their ability to effectively present soluble antigens. This functional loss was associated with alterations in the expression of adhesion molecules and CD14 and a reduction in the uptake of soluble antigen. Recently, we have demonstrated that Salmonella typhi flagella (STF) markedly decrease CD14 expression and are potent inducers of proinflammatory cytokine production by human peripheral blood mononuclear cells (hPBMC). In order to determine whether S. typhi and soluble STF also alter the ability of Mφ to activate T cells to proliferate to antigens and mitogens, hPBMC were cultured in the presence of tetanus toxoid (TT) or phytohemagglutinin (PHA) and either killed whole-cell S. typhi or purified STF protein. Both whole-cell S. typhi and STF suppressed proliferation to PHA and TT. This decreased proliferation was not a result of increased Mφ production of nitric oxide, prostaglandin E 2 , or oxygen radicals or the release of interleukin-1β, tumor necrosis factor alpha, interleukin-6, or interleukin-10 following exposure to STF. However, the ability to take up soluble antigen, as determined by fluorescein isothiocyanate-labeled dextran uptake, was reduced in cells cultured with STF. Moreover, there was a dramatic reduction in the expression of CD54 on Mφ after exposure to STF. These results indicate that whole-cell S. typhi and STF have the ability to alter in vitro proliferation to soluble antigens and mitogens by affecting Mφ function.

Many immunosuppressive effects of Trypanosoma cruzi can be reproduced in vitro by a preparation consisting of molecules spontaneously released by this protozoan (termed trypanosomal immunosuppressive factor (TIF)). In this work, we attempted to establish whether TIF-induced inhibition of lymphoproliferation results from preventing lymphocyte activation or impairing a post-activation process. Although [3H]thymidine uptake and expression of CD25 by normal human T lymphocytes stimulated with a phorbol ester were markedly reduced by T. cruzi or TIF, translocation of cytosolic protein kinase C (PKC) to the cell membrane was not affected. Lymphoproliferation induced by ionomycin was also inhibited by T. cruzi or TIF but the typical elevation of intracellular calcium ions [Ca2+]i caused by this calcium ionophore was not altered. The increase in [Ca2+]i induced with anti-CD3 antibody was also unaffected by TIF. TIF did not preclude lymphocytes stimulated with phytohemagglutinin from accumulating normal mRNA levels of NFAT1 (also known as NFATp) and NFATc. NFAT1 and NFATc are components of the NFAT complex that controls transcription of genes coding for several cytokines and whose translocation to the nucleus is dependent upon PKC activation and increased [Ca2+]i. In contrast, the mRNA levels of cyclin D2 and cdk4, which form a holoenzyme complex known to regulate cell progression through the G1 phase, were markedly reduced by TIF. These results indicated that TIF did not inhibit lymphocyte activation leading to early secondary signaling but curtailed a mechanism controlling cell progression through G1 and necessary for reaching S phase.

Summary A substantial proportion of human peripheral blood mononuclear cells (PBMC) manifested a decreased capacity to express membrane interferon-γ receptors (IFN-γR) when co-cultured with Trypanosoma cruzi. Among the lymphocytes, B cells accounted for the bulk of this effect, evidenced by a marked drop in the proportion of CD19+ or CD20+ cells expressing IFN-γR. Decreased IFN-γR expression by B lymphocytes was seen as early as 3 h after co-culture with T. cruzi and persisted for at least 24 h. The parasite had no detectable effect on CD19, CD20 or DR antigen expression by B lymphocytes. Neither the proportion ofB cells expressing these markers nor the membrane density of these molecules varied significantly in the presence of T. cruzi. In PBMC cultures stimulated with Staphlyococcus aureus Cowan I (SACI), T. cruzi decreased the percentages of both IFN-γR+ and IFN-R +bnght (cells expressing above-normal levels of surface IFN-γR) B lymphocytes. Cell-free filtrates of T. cruzi suspensions reproduced the suppressive effects of living parasites on IFN-γR expression by B cells. When T. cruzi was present, the intracellular levels of IFN-γR molecules in resting or SACI-activated B lymphocytes, represented by fluorescence intensity, were well below control values, suggesting that decreased surface expression resulted from suppressed IFN-γR synthesis. Among T (CD3+) cells, 10–8% to 39–6% (7 donors) expressed surface IFN-γR and did so at a very low level. These percentages were also reduced by T. cruzi. If occurring in the host, downregulated expression of IFN-γR could curtail the utilization of IFN-γ, known to play a critical role in host defence against T. cruzi infection.

We evaluated the relative contribution of the humoral and cellular arms of the immune response to bone marrow cells transplanted into sensitized recipients. We report here for the first time that humoral immunity contributes predominantly to allosensitization. Although the major role for nonmyeloablative conditioning is to control alloreactive host T cells in nonsensitized recipients, strikingly, none of the strategies directed primarily at T-cell alloreactivity enhanced engraftment in sensitized mice. In evaluating the mechanism behind this barrier, we found that humoral immunity plays a critical role in the rejection of allogeneic marrow in sensitized recipients. Adoptive transfer of as little as 25 μL serum from sensitized mice abrogated engraftment in secondary naive recipients. With the use of μMT mice as recipients, we found that T-cell-mediated immunity plays a secondary but still significant role in allorejection. Targeting of T cells in sensitized B-cell-deficient μMT mice enhanced alloengraftment. Moreover, both T- and B-cell tolerance were achieved in sensitized recipients when allochimerism was established, as evidenced by the acceptance of second donor skin grafts and loss of circulating donor-specific Abs. These findings have important implications for the management of sensitized transplant recipients and for xenotransplantation in which B-cell reactivity is a predominant barrier.

Type 1 diabetes is a systemic autoimmune disease that can be cured by transplantation of hematopoietic stem cells (HSCs) from disease-resistant donors. Nonobese diabetic (NOD) mice have a number of features that distinguish them as bone marrow transplant recipients that must be understood prior to the clinical application of chimerism to induce tolerance. In the present studies, we characterized NOD HSCs, comparing their engraftment characteristics to HSCs from disease-resistant strains. Strikingly, NOD HSCs are significantly enhanced in engraftment potential compared with HSCs from disease-resistant donors. Unlike HSCs from disease-resistant strains, they do not require graft-facilitating cells to engraft in allogeneic recipients. Additionally, they exhibit a competitive advantage when coadministered with increasing numbers of syngeneic HSCs, produce significantly more spleen colony-forming units (CFU-Ss) in vivo in allogeneic recipients, and more granulocyte macrophage–colony-forming units (CFU-GMs) in vitro compared with HSCs from disease-resistant controls. NOD HSCs also exhibit significantly enhanced chemotaxis to a stromal cell–derived factor 1 (SDF-1) gradient and adhere significantly better on primary stroma. This enhanced engraftment potential maps to the insulin-dependent diabetes locus 9 (Idd9) locus, and as such the tumor necrosis factor (TNF) receptor family as well as ski/sno genes may be involved in the mechanism underlying the autonomy of NOD HSCs. These findings may have important implications to understand the evolution of autoimmune disease and impact on potential strategies for cure.

In the present study we examined the effect of the timing of marrow infusion on engraftment in nonmyeloablatively conditioned mice.B10 mice were conditioned with decreasing doses of total body irradiation (TBI) and reconstituted with bone marrow cells (BMCs) from major histocompatibility complex-disparate donor B10.BR mice at 0 or 6 hr, or on days 1, 2, 3, 4, 5, 8, and 12 with respect to TBI.After undergoing conditioning with 700 cGy TBI and transplantation with 15 x 10(6) BMCs, 100% of recipients engrafted if the marrow was infused between 0 and 4 days after TBI. For lower doses of TBI, a delay in infusion of the marrow after TBI conditioning was associated with a significant increase in engraftment. Significantly less engraftment was achieved in animals conditioned with 600 cGy TBI if the marrow was infused at 0 or 6 hr compared with a 1- to 4-day delay. When the TBI was decreased to 500 cGy, engraftment occurred only when the transplant was performed between days 2 and 8. The highest proportion of recipients engrafted when the marrow was infused on day 4. This enhanced engraftment after a delay in marrow infusion is associated with a significant reduction in host mixed lymphocyte reaction reactivity and is correlated inversely with serum levels of interleukin-6 in the recipient.These data demonstrate for the first time that a delay between conditioning and marrow infusion significantly improves allogeneic engraftment in nonmyeloablatively conditioned recipients and reduces the total conditioning required.

Immunosuppression by the fungal metabolite cyclosporin A (CsA) is characterized by functional inhibition, rather than destruction of cells. Because activation of immune cells involves intracellular signalling events associated with modulations of cell transmembrane potential (TMP), we tested the ability of cyclosporin A (CsA) to modulate immune mononuclear cell TMP in vitro using a TMP sensitive cationic dye, dihexyloxacarbocyanine (DIOC6(3)). All analyses were performed by flow cytometry. CsA increased TMP in monocytes and lymphocytes isolated from the blood of healthy human volunteers. CsA-induced hyperpolarization was time and concentration dependent in monocytes while the lymphocyte hyperpolarization, although time dependent, was evident over the entire range of CsA concentrations tested. CsA-induced hyperpolarization of lymphocytes was dependent on potassium ion (K+) efflux as indicated by the absence of hyperpolarization in 154 mM KCl or with pretreatment with 100 uMM quinine (an inhibitor of K+ channels). Monocyte hyperpolarization by CsA was not inhibited in either system. Dihydrocyclosporin C (DH-CsC), an immunosuppressive analog of CsA, also hyperpolarized mononuclear cells. The anionic TMP sensitive dye bis oxonol (di-BA-C4) indicated that CsA treatment depolarized mononuclear cell plasma membranes. The mitochondrial poison carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazine (FCCP) eliminated CsA induced hyperpolarization and also indicated that CsA caused plasma membrane depolarization. We conclude that brief in vitro exposure to cyclosporin alters the transmembrane electrical potential of human lymphocytes and monocytes. © 1993 Wiley-Liss, Inc.

CD8+/TCR− facilitating cells (FC) enhance engraftment of purified hematopoietic stem cells (HSC) in syngeneic and allogeneic recipients. FC also induce the production of regulatory T cells (Treg) in vivo and in vitro. The B220+/CD11c+/CD11b− precursor plasmacytoid dendritic cell (p-preDC) subpopulation in FC (p-preDC FC) is critical to FC function. However, p-preDC FC are significantly less efficient in function compared to FC total. In this study, we evaluated the phenotype and function of FC from diabetes-prone nonobese diabetes (NOD) mice. We found that NOD FC contain subpopulations similar to those previously described in B6 FC, including p-preDC, CD19+, NK1.1+DX5+ and myeloid cells. P-preDC represent the major FC subpopulation in NOD mice. The CD19+, DX5+ and B220−/CD11c+/CD11b+ subpopulations were significantly decreased in NOD FC compared to those from B6 or NOR mice (Figure 1a; * = P< 0.05; ** = P< 0.007). To test the function of NOD FC, 500 HSC (c-Kit+/Sca-1+/Lin−) were sorted and transplanted with or without 30,000 FC into conditioned 950 cGy recipients. MHC-matched diabetes-resistant NOR mice were served as a control strain for NOD mice. 5 (31%) of 16 recipients of NOR HSC were engrafted and survived up to 130 days. 7 (70%) of 10 recipients of NOR HSC plus FC have long-term engraftment and survival over 130 days, indicating that the NOR FC significantly enhanced engraftment of NOR HSC compared to HSC alone. In striking contrast, NOD FC were functionally impaired and did not enhance HSC engraftment in NOD recipients as evidenced by similar engraftment of HSC with FC (31%, n = 13) compared to the HSC alone (24%, n = 17; P = 0.579). Notably, when NOD mice were treated with Flt3 ligand (FL; 10 μg/ subcutaneous/daily, 10 days), FC were expanded in peripheral blood (PB). The DX5+ and B220−/CD11c+/CD11b+ subpopulations were significantly increased (Figure 1b). The FL-PB FC significantly facilitate engraftment of allogeneic HSC in vivo in the NOD → B10 model (Figure 1c; P = 0.02). These data demonstrate that NOD FC exhibit significantly impaired function and FL plays an important role in regulation and development of FC function. We propose that the defective function of NOD FC is mechanistically due either to the abnormal activation status of the p-preDC FC population and/or the absence of collaborative subpopulations in FC such as NK FC. This hypothesis offers an attractive explanation for the mechanism of FC to enhance HSC engraftment and to induce tolerance. Studies are underway to evaluate the mechanism by which FC contribute to diabetes-pathogenesis and/or prevention. Figure Figure

Recipient sensitization to MHC antigens from transfusion therapy and prior graft rejection is among the most critical of problems in clinical transplantation. Sensitized patients reject vascularized organ or bone marrow transplants within minutes to hours as a result of preformed anti-donor Abs. Preventing allosensitization at the time recipients are exposed to donor alloantigens would be of obvious clinical benefit. B cell activation and the generation of memory B cells depends upon T cell responses via signaling from the co-stimulatory molecule CD154 (on activated T cells) to CD40 (on B cells). We have demonstrated in an allogeneic mouse model [BALB/c (H2Kd) to B6 (H2Kb)] that blockade of T and B cell interactions with anti-CD154 induces B cell tolerance, as defined by complete abrogation of the generation of donor-specific Ab after skin grafting. Furthermore, anti-CD154 treatment promotes successful subsequent bone marrow transplantation in these recipients, confirming that sensitization was prevented. In this study, we evaluated the effect of anti-CD154 mAb on T- and B-cell populations, activation state, and cytokine expression by T cells. B6 recipients were treated with anti-CD154 (day 0 and +3) or isotype hamster IgG control around the time receiving BALB/c skin grafts (day 0), and the number of T-cells (CD4+ and CD8+), total B-cells (CD19+), immature B-cells (CD19+CD24highCD23low), and follicular B-cells (CD19+CD24lowCD23high) in the spleen was enumerated by 4 color flow cytometry at day 7, 15 and 25 after skin grafting. No significant difference in absolute number of T- and B-cell subpopulations was seen between anti-CD154 and control IgG treated groups at the time points tested. By measuring the percentage CD71+ cells in the CD8+ or CD4+ gate or CD69+ in the CD19+ gate, activated T and B cell populations were evaluated. In vivo blockade of CD154 resulted in a significantly reduced activation of alloreactive T- and B-cells: the percentage of CD8+/CD71+ T cells was significantly lower at day 7 and the percentage of CD4+/CD71+ T cells was significantly lower at all time points compared with control mice (P < 0.05). The percentage of CD19+/CD69+ B cells at day 7 and 25 was significantly lower compared with control IgG treated mice (P < 0.05). To determine the effect of anti-CD154 treatment on Th1 and Th2 cytokine production, intracellular IFN-γ and IL-10 expression was analyzed. The IFN-γ expression in both CD8 and CD4 T-cells was inhibited at day 7 and reached significance (P < 0.01) by day 15 compared with control IgG treated group. IL-10, a cytokine which promotes B-cell activation and differentiation expression, was similar at day 7 between the two groups, but significantly decreased in both CD8 and CD4 T-cells at day 15 in mice treated with anti-CD154. Therefore, these data suggest that blockade of CD154 during initial antigen exposure mechanistically interferes with activation of both allo antigen-specific T and B-cells and inhibits the generation of allogeneic Ab (allosensitization). These effects are associated with suppression of IFN-γ and IL-10 cytokine secretion. Figure Figure

Allosensitization resulting from transfusion therapy is a major challenge for the use of bone marrow transplantation to treat sickle cell disease. Prior exposure to foreign major histocompatibility complex (MHC) antigens through transfusion or transplantation is associated with an increased rate of solid organ graft rejection. Using a mouse model for sensitization, we recently found that humoral immunity plays a dominant role in sensitization to MHC alloantigens and concomitant graft rejection. The relative contribution of cellular versus humoral adaptive immune responses has not been fully characterized. In this study, we explored the role of host T cells in rejection of allografts by sensitized recipients using μ MT mice which are defective in producing mature B cells and antibody. Therefore, the barrier from preformed antibodies in sensitized normal mice would not exist in μ MT mice. μ MT (H-2b) mice were sensitized with BALB/c (H-2d) skin grafts. Although no anti-MHC alloantibody was detected in the μ MT mice after rejection, the skin grafts were rejected with a kinetic similar to normal controls (MST = 14.1 ± 1.2 days versus 13.6 ± 1.5 days). The prompt rejection of skin allografts by B cell deficient mice suggests that T cell activation and function are normal in vivo and that T cells alone are sufficient to reject allogeneic skin grafts. BMT was subsequently performed in presensitized μ MT mice 5 weeks after sensitization as well as in naïve μ MT mice. All naïve μ MT mice (n = 6) engrafted with 700 cGy TBI and 30 x 106 bone marrow cells, but none of the presensitized μ MT mice engrafted. Since humoral immunity is absent in sensitized μ MT mice, the increased barrier in these mice therefore must be mediated by the primed T cells. To further define the relative contribution of T cell subpopulations to alloresistance, we targeted these populations in vivo as preconditioning using monoclonal antibodies (mAb). Sensitized μ MT mice were treated with anti-α β-TCR, anti-CD8, anti-CD4 or anti-CD154 mAbs alone or in combination and 850 cGy of total body irradiation, then transplanted with untreated BALB/c donor bone marrow cells (Figure). Engraftment did not occur in the sensitized μ MT mice without mAb treatment, while nearly all animals engrafted with preconditioning with anti-α β-TCR alone, anti-CD8 plus anti-CD154, or anti-CD8 plus anti-CD4 (100%, 87.5%, and 100% respectively) and transplantation with 30 x 106 donor BM cells. Moreover, anti-α β-TCR preconditioning promoted engraftment in all sensitized μ MT mice with transplantation of as low as 15 x 106 BM cells. These data demonstrate that T cell mediated cellular immunity contributes to rejection in sensitized recipients, and that successful therapies to achieve allogeneic engraftment using nonmyeloablative conditioning in sensitized recipients will need to address both arms of the adaptive antigen-specific immune system: cellular and humoral. Figure Figure

Facilitating cells (FC) are a CD8+/TCR− subpopulation of bone marrow (BM) cells that enhance engraftment of hematopoietic stem cells (HSC) in allogeneic recipients without causing graft-versus-host disease. They also significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. Treatment of mice with Flt3 ligand (Flt3-L) induces significant expansion of FC and HSC in the BM and their mobilization into peripheral blood (PB). In the present study, we evaluated the function of Flt3-L expanded FC and potential mechanism of action. Flt3-L treatment resulted in an 8.5-fold increase of FC in BM and a 100-fold increase in PB compared to untreated mice. When FC from the PB and BM of Flt3-L-treated donors were transplanted with HSC into allogeneic recipient mice, BM FC exhibited significantly impaired function while PB FC were potently functional (Fig. a). Strikingly, this correlated with an increase in mRNA for the chemokine SDF-1 and its receptor, CXCR4, which were significantly increased in PB FC (≥10 fold) and decreased in Flt3-L-expanded BM FC as compared to untreated control BM FC (Fig. b). The impaired function for BM FC was restored within 5 days after cessation of Flt3-L-treatment. Taken together, these data suggest that FC can be mobilized efficiently with Flt3-L and their engraftment facilitating function is associated with upregulation of transcripts for SDF-1 and CXCR4, suggesting that FC may chemoattract HSC by secreting SDF-1 and enhance their homing to the new microenvironment after transplantation. The fact that FC express CXCR4 suggests that they may co-migrate with HSC to the hematopoietic microenvironment after transplantation, being chemoattracted by BM-derived SDF-1. In summary, we provide evidence that FC are mobilized with HSC; the SDF-1-CXCR4 axis is involved in FC:HSC interaction; and FC co-migrate with HSC in response to an SDF-1 gradient. Figure Figure

Approaches to enhance engraftment of hematopoietic stem cells (HSC) are of primary interest in BM transplantation. CD8+TCR− facilitating cells (FC) improve HSC engraftment in allogeneic recipients without causing graft versus host disease. FC also significantly enhance the engraftment of limiting numbers of HSC in syngeneic recipients, suggesting that FC act directly on HSC. We therefore analyzed the mechanism for FC-mediated effects on HSC. We found that FC increased the ability of purified HSC to generate colonies in both the CFC and CAFC/LTC-IC assays, confirming a direct effect of FC on different HSC compartments. Co-incubation of HSC with FC for 18 or 40 hours significantly increased the survival of HSC and their subsequent ability to generate CFC at these same time points. We determined that the anti-apoptotic effect of FC on HSC was associated with the up regulation of anti-apoptotic Bcl-3 transcripts. We postulated here that the effect of FC on HSC was due to cytokine secretion. As FC produce TNFα after CpG ODN stimulation and TNFα has various activities on HSC, we evaluated the role of TNFα on FC function. FC were sorted from TNFα deficient mice and the facilitative activity of FC on HSC engraftment was assessed. FC from TNFα deficient mice were impaired in facilitating HSC engraftment in both the syngeneic and allogeneic models, suggesting a role for TNFα in FC function. Notably, TNFα transcripts were present in FC by 16 hours of co-incubation of FC + HSC and FC produce TNFα (surface and intra-cellular) when in contact with HSC. Furthermore, when TNFα was blocked (using anti-TNFα mAb), FC from wild type mice lost the ability to increase HSC clonogenicity (from 38.2±13.6 CFC/1000 HSC in HSC alone to 65.7 ± 22 for HSC + FC and 38.2 ± 19.6 for FC pre-incubated 1 hour with anti-TNFα mAb before incubation with HSC). Moreover, anti-TNFα mAb also blocked the ability of FC to up-regulate Bcl-3 transcripts in HSC. In conclusion, FC act directly on HSC via several mechanisms to maintain the balance between proliferation/differentiation/survival of HSC. One central mechanism implicates TNFα production by FC, which may protect HSC from undergoing apoptosis by up-regulating anti-apoptotic transcripts (e.g. Bcl-3). These findings may have great impact for the use of accessory cells in HSC transplantation, especially when numbers of HSC are limiting.

Multiple hematopoietic defects have been defined in NOD mice and in humans with type I diabetes, including defects in myeloid cells and antigen presenting cells that correlate with diabetes progression. Since the replacement of HSC in NOD mice can eliminate the progression of autoimmunity and control on-going autoimmune responses, we characterized the function of HSC from NOD mice. We found that purified HSC from NOD mice have an autonomous behavior when transplanted in allogeneic recipient strains as reflected by significantly enhanced engraftment in allogeneic recipients. NOD HSC were able to compete for engraftment with syngeneic HSC even when the NOD and syngeneic HSC were given at a 1:1 ratio. NOD BMC produced a higher number of splenic colonies compared to B10.BR BMC in the allogeneic day 12 CFU-S assay. We also demonstrated that NOD HSC had a high resistance to irradiation, as reflected by the cell survival 20 hours after irradiation and in the in vitro CFC assay. These data suggest that NOD HSC escape alloreactivity and compete with normal HSC. The enhanced engraftment ability in allogeneic recipients of NOD HSC was not due to an increase in frequency of primitive HSC, enumerated by day 35 cobblestone area forming cells (CAFC). This finding was further confirmed by the fact that there was no difference in the long-term repopulating cell phenotype (CD49e+/CD49ddim) between HSC obtained from NOD, B10.BR or C57BL/10 mice. Notably, NOD bone marrow cells exhibit significantly enhanced chemotaxis to SDF-1 in vitro and significantly increased HSC adhesion to primary stroma. This was associated with an increase in the expression of VCAM-1, ICAM-1 and ICAM-2 on NOD HSC. Using NOD mice congenic at selected Idd loci with C57BL/10, we determined that the enhanced engraftment potential of NOD HSC mapped to the Idd9 (insulin-dependent diabetes) locus and, as such, the TNF receptor family as well as ski/sno genes may be involved in the mechanism underlying the autonomy of NOD HSC. In conclusion, NOD HSC exhibit increased autonomy in vivo and in vitro compared to non-diabetic strains, and engraft better in allogeneic recipients, possibly due to enhanced migration and adherence to the microenvironment. This finding may be of interest for a better understanding of disease pathogenesis and in developing cell-based strategies to cure diabetes.