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David L Sacks

Publications and source records attributed to David L Sacks.

17 recordsLinked to original sources
PubMed2006

A Leishmania amazonensis ZIP family iron transporter is essential for parasite replication within macrophage phagolysosomes.

Infection of mammalian hosts with Leishmania amazonensis depends on the remarkable ability of these parasites to replicate within macrophage phagolysosomes. A critical adaptation for survival in this harsh environment is an efficient mechanism for gaining access to iron. In this study, we identify and characterize LIT1, a novel L. amazonensis membrane protein with extensive similarity to IRT1, a ZIP family ferrous iron transporter from Arabidopsis thaliana. The ability of LIT1 to promote iron transport was demonstrated after expression in yeast and in L. amazonensis LIT1-null amastigotes. Endogenous LIT1 was only detectable in amastigotes replicating intracellularly, and its intracellular expression was accelerated under conditions predicted to result in iron deprivation. Although L. amazonensis lacking LIT1 grew normally in axenic culture and had no defects differentiating into infective forms, replication within macrophages was abolished. Consistent with an essential role for LIT1 in intracellular growth as amastigotes, Deltalit1 parasites were avirulent. After inoculation into highly susceptible mice, no lesions were detected, even after extensive periods of time. Despite the absence of pathology, viable Deltalit1 parasites were recovered from the original sites of inoculation, indicating that L. amazonensis can persist in vivo independently of the ability to grow in macrophages. Our findings highlight the essential role played by intracellular iron acquisition in Leishmania virulence and identify this pathway as a promising target for therapeutic intervention.

Amino Acid Sequence↗
PubMed2005

Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice.

Experimental Leishmania major infection in mice has been of immense interest because it was among the first models to demonstrate the importance of the Th1/Th2 balance to infection outcome in vivo. However, the Th2 polarization that promotes the development of nonhealing cutaneous lesions in BALB/c mice has failed to adequately explain the mechanisms underlying nonhealing forms of leishmaniasis in humans. We have studied a L. major strain from a patient with nonhealing lesions that also produces nonhealing lesions with ulcerations and high parasite burden in conventionally resistant C57BL/6 mice. Surprisingly, these mice develop a strong, polarized, and sustained Th1 response, as evidenced by high levels of IFN-gamma produced by Leishmania-specific cells in the draining lymph node and in the ear lesion, and an absence of IL-4 or IL-13. The parasites fail to be effectively cleared despite high level induction of inducible NO synthase in the lesion, and despite their sensitivity to killing by IFN-gamma-activated macrophages in vitro. Infection of IL-10(-/-) mice, blockade of the IL-10R, or depletion of CD25(+) cells during the chronic phase promotes parasite killing, indicating that IL-10 and regulatory T cells play a role in rendering the Th1 responses ineffective at controlling infection in the skin. Mice with nonhealing primary lesions are nonetheless resistant to reinfection in the other ear. We suggest that nonhealing infections in animal models that are explained not by aberrant Th2 development, but by overactivation of homeostatic pathways designed to control inflammation, provide better models to understand nonhealing or reactivation forms of leishmaniasis in humans.

Animals↗
PubMed2005

Antigen requirements for efficient priming of CD8+ T cells by Leishmania major-infected dendritic cells.

CD4(+) and CD8(+) T-cell responses have been shown to be critical for the development and maintenance of acquired resistance to infections with the protozoan parasite Leishmania major. Monitoring the development of immunodominant or clonally restricted T-cell subsets in response to infection has been difficult, however, due to the paucity of known epitopes. We have analyzed the potential of L. major transgenic parasites, expressing the model antigen ovalbumin (OVA), to be presented by antigen-presenting cells to OVA-specific OT-II CD4(+) or OT-I CD8(+) T cells. Truncated OVA was expressed in L. major as part of a secreted or nonsecreted chimeric protein with L. donovani 3' nucleotidase (NT-OVA). Dendritic cells (DC) but not macrophages infected with L. major that secreted NT-OVA could prime OT-I T cells to proliferate and release gamma interferon. A diminished T-cell response was observed when DC were infected with parasites expressing nonsecreted NT-OVA or with heat-killed parasites. Inoculation of mice with transgenic parasites elicited the proliferation of adoptively transferred OT-I T cells and their recruitment to the site of infection in the skin. Together, these results demonstrate the possibility of targeting heterologous antigens to specific cellular compartments in L. major and suggest that proteins secreted or released by L. major in infected DC are a major source of peptides for the generation of parasite-specific CD8(+) T cells. The ability of L. major transgenic parasites to activate OT-I CD8(+) T cells in vivo will permit the analysis of parasite-driven T-cell expansion, differentiation, and recruitment at the clonal level.

Animals↗
PubMed2005

Conditions influencing the efficacy of vaccination with live organisms against Leishmania major infection.

Numerous experimental vaccines have been developed with the goal of generating long-term cell-mediated immunity to the obligate intracellular parasite Leishmania major, yet inoculation with live, wild-type L. major remains the only successful vaccine in humans. We examined the expression of immunity at the site of secondary, low-dose challenge in the ear dermis to determine the kinetics of parasite clearance and the early events associated with the protection conferred by vaccination with live L. major organisms in C57BL/6 mice. Particular attention was given to the route of vaccination. We observed that the rapidity, strength, and durability of the memory response following subcutaneous vaccination with live parasites in the footpad are even greater than previously appreciated. Antigen-specific gamma interferon (IFN-gamma)-producing T cells infiltrate the secondary site by 1.5 weeks, and viable parasites are cleared as early as 2.5 weeks following rechallenge, followed by a rapid drop in IFN-gamma(+) CD4(+) cell numbers in the site. In comparison, intradermal vaccination with live parasites in the ear generates immunity that is delayed in effector cell recruitment to the rechallenge site and in the clearance of parasites from the site. This compromised immunity was associated with a rapid recruitment of interleukin-10 (IL-10)-producing CD4(+) T cells to the rechallenge site. Treatment with anti-IL-10-receptor or anti-CD25 antibody enhanced early parasite clearance in ear-vaccinated mice, indicating that chronic infection in the skin generates a population of regulatory cells capable of influencing the level of resistance to reinfection. A delicate balance of effector and regulatory T cells may be required to optimize the potency and durability of vaccines against Leishmaniasis and other intracellular pathogens.

Animals↗
PubMed2005

Prevention and treatment of cutaneous leishmaniasis in primates by using synthetic type D/A oligodeoxynucleotides expressing CpG motifs.

Oligodeoxynucleotides (ODN) containing CpG motifs mimic microbial DNA and are recognized by toll-like receptor 9 on immune cells. The resulting response limits the early spread of infectious organisms and promotes the development of adaptive immunity. In this regard, CpG ODN show promise as immunoprotective agents and as vaccine adjuvants. Previous studies of nonhuman primates showed that administration of CpG ODN type D (also known as type A) at the site of infection 3 days before and after a challenge with Leishmania major enhanced host resistance and reduced the lesion's severity. In this study, we show that systemic administration of D/A ODN limits the size of lesions following an intradermal infection with L. major. Importantly, the reduced morbidity was not associated with a reduction in long-term immunity, as such treated macaques were still protected following a secondary challenge. Finally, administration of D/A ODN to macaques that had established cutaneous lesions reduced the severity of the lesions, suggesting a potential role for CpG ODN in L. major treatment. Together, these findings support the development of clinical studies to assess the use of CpG ODN types D/A as immunoprotective and therapeutic agents.

Animals↗
PubMed2004

A role for insect galectins in parasite survival.

Insect galectins are associated with embryonic development or immunity against pathogens. Here, we show that they can be exploited by parasites for survival in their insect hosts. PpGalec, a tandem repeat galectin expressed in the midgut of the sandfly Phlebotomus papatasi, is used by Leishmania major as a receptor for mediating specific binding to the insect midgut, an event crucial for parasite survival, and accounts for species-specific vector competence for the most widely distributed form of cutaneous leishmaniasis in the Old World. In addition, these studies demonstrate the feasibility of using midgut receptors for parasite ligands as target antigens for transmission-blocking vaccines.

Amino Acid Sequence↗
PubMed2003

Persistence without pathology in phosphoglycan-deficient Leishmania major.

Leishmania infections involve an acute phase of replication within macrophages, typically associated with pathology. After recovery parasites persist for long periods, which can lead to severe disease upon reactivation. Unlike the role of host factors, parasite factors affecting persistence are poorly understood. Leishmania major lacking phosphoglycans (lpg2-) were unable to survive in sand flies and macrophages, but retained the ability to persist indefinitely in the mammalian host without inducing disease. The L. major lpg2- thus provides a platform for probing parasite factors implicated in persistence and its role in disease and immunity.

Animals↗
PubMed2003

Comparison of the A2 gene locus in Leishmania donovani and Leishmania major and its control over cutaneous infection.

In Old World Leishmania infections, Leishmania donovani is responsible for fatal visceral leishmaniasis, and L. major is responsible for non-fatal cutaneous leishmaniasis in humans. The genetic differences between these species which govern the pathology or site of infection are not known. We have therefore carried out detailed analysis of the A2 loci in L. major and L. donovani because A2 is expressed in L. donovani but not L. major, and A2 is required for survival in visceral organs by L. donovani. We demonstrate that although L. major contains A2 gene regulatory sequences, the multiple repeats that exist in L. donovani A2 protein coding regions are absent in L. major, and the remaining corresponding A2 sequences appear to represent non-expressed pseudogenes. It was possible to restore amastigote-specific A2 expression to L. major, confirming that A2 regulatory sequences remain functional in L. major. Although L. major is a cutaneous parasite in rodents and humans, restoring A2 expression to L. major inhibited its ability to establish a cutaneous infection in susceptible BALB/c or resistant C57BL6 mice, a phenotype typical of L. donovani. There was no detectable cellular immune response against L. major after cutaneous infection with A2-expressing L. major, suggesting that the lack of growth was not attributable to acquired host resistance but to an A2-mediated suppression of parasite survival in skin macrophages. These observations argue that the lack of A2 expression in L. major contributed to its divergence from L. donovani with respect to the pathology of infection.

3' Untranslated Regions↗
PubMed2003

The relative contribution of IL-4 receptor signaling and IL-10 to susceptibility to Leishmania major.

The roles of IL-10 and IL-4 receptor signaling were evaluated in a murine model of Leishmania major infection. In previous studies the L. major substrain LV39 caused progressive, nonhealing lesions in BALB/c mice deficient for IL-4R alpha-chain (IL-4R alpha), while substrain IR173 was highly controlled. To explore whether IL-10 is responsible for inducing susceptibility to LV39, wild-type and IL-4R alpha(-/-) mice were treated with anti-IL-10R mAb, and in a genetic approach, the IL-4R alpha(-/-) mice were crossed with BALB/c IL-10(-/-) mice. In contrast to the lack of resistance conferred by IL-4R alpha gene deletion, partial resistance to LV39 was conferred by IL-10 gene deletion or treatment of BALB/c mice with anti-IL-10R mAb. Lesion sizes and LV39 parasite numbers were further and dramatically reduced in both anti-IL-10R-treated IL-4R alpha(-/-) mice and IL-4R alpha x IL-10 double knockouts. Anti-IL-10R mAb treatment further suppressed parasite growth in IL-4R alpha(-/-) mice infected with L. major IR173. Production of IFN-gamma was only increased relative to wild-type or littermate controls in IL-4R alpha(-/-) mice with complementary defects in IL-10. Comparisons of IFN-gamma-treated infected macrophages in vitro indicated that LV39 required 25- to 500-fold greater concentrations of IFN-gamma than IR173-infected macrophages to achieve a similar efficiency of parasite killing. These studies suggest that regardless of parasite substrain, IL-10 is as important as IL-4/IL-13 in promoting susceptibility to L. major and even more so for those substrains that are relatively resistant to IFN-gamma mediated killing.

Animals↗
PubMed2003

Coinjection with CpG-containing immunostimulatory oligodeoxynucleotides reduces the pathogenicity of a live vaccine against cutaneous Leishmaniasis but maintains its potency and durability.

The inoculation of live, nonattenuated Leishmania major to produce a lesion in a selected site that heals, referred to as leishmanization, is to date the only vaccine against leishmaniasis that has proven to be effective in humans. Its use has been restricted or abandoned entirely, however, due to safety concerns. In an attempt to develop a leishmanization protocol that minimizes pathology while maintaining long-term protection, live parasites were coinjected with CpG-containing immunostimulatory oligodeoxynucleotides (CpG ODNs) alone or in combination with whole-cell lysates of heat-killed L. major promastigotes bound to alum (ALM). C57BL/6 mice infected intradermally by using L. major plus CpG ODN with or without ALM developed few or no dermal lesions and showed an early containment of parasite growth, while mice infected with L. major with or without ALM developed sizable dermal lesions that required up to 10 weeks to heal. The CpG ODNs provoked a transient inflammation that included an early recruitment and accumulation of gamma interferon-producing CD4(+) lymphocytes in the site. Attenuation of the live vaccine did not compromise its ability to confer long-term immunity, as mice receiving L. major and CpG ODN plus ALM were totally protected against reinfection with L. major for up to 6 months. By comparison, the immunity elicited by two efficient nonlive vaccines began to wane by 6 months. Our results suggest that immune modulation using CpG ODNs might be a practical approach to improving the safety of a highly effective live vaccine that has already been widely applied.

Adjuvants, Immunologic↗
PubMed2002

CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity.

The long-term persistence of pathogens in a host that is also able to maintain strong resistance to reinfection, referred to as concomitant immunity, is a hallmark of certain infectious diseases, including tuberculosis and leishmaniasis. The ability of pathogens to establish latency in immune individuals often has severe consequences for disease reactivation. Here we show that the persistence of Leishmania major in the skin after healing in resistant C57BL/6 mice is controlled by an endogenous population of CD4+CD25+ regulatory T cells. These cells constitute 5-10% of peripheral CD4+ T cells in naive mice and humans, and suppress several potentially pathogenic responses in vivo, particularly T-cell responses directed against self-antigens. During infection by L. major, CD4+CD25+ T cells accumulate in the dermis, where they suppress-by both interleukin-10-dependent and interleukin-10-independent mechanisms-the ability of CD4+CD25- effector T cells to eliminate the parasite from the site. The sterilizing immunity achieved in mice with impaired IL-10 activity is followed by the loss of immunity to reinfection, indicating that the equilibrium established between effector and regulatory T cells in sites of chronic infection might reflect both parasite and host survival strategies.

Animals↗
PubMed2002

Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against leishmania major infection.

CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major-specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.

Animals↗
PubMed2002

Targeted gene deletion in Leishmania major identifies leishmanolysin (GP63) as a virulence factor.

Leishmanolysin, the Leishmania surface metalloproteinase of 63 kDa (GP63) has been described as a parasite virulence factor and is involved in the direct interaction of promastigotes and host macrophage receptors and interaction with the complement cascade. To study the role of leishmanolysin in the pathogenesis and virulence of Leishmania major, targeted gene replacement was used to delete the entire 20 kb region containing all seven leishmanolysin genes (gp63 genes 1-7). The resulting L. major leishmanolysin deficient mutants showed normal development inside the sand fly vector, however, promastigotes recovered from sand flies or from culture showed an increase in sensitivity to complement-mediated lysis and a delay in lesion formation in BALB/c animals. The phenotypic differences could be significantly improved by expression of a cloned leishmanolysin gene. These results demonstrate that leishmanolysin is a vital virulence factor in Leishmania pathogenesis.

Animals↗
PubMed2002

Skin-derived macrophages from Leishmania major-susceptible mice exhibit interleukin-12- and interferon-gamma-independent nitric oxide production and parasite killing after treatment with immunostimulatory DNA.

Co-administration of CpG-containing immunostimulatory oligodeoxynucleotides and parasite antigen protects susceptible BALB/c mice from otherwise progressive infection with Leishmania major. Although the protective effect of CpG-containing immunostimulatory oligodeoxynucleotides is clearly dependent on endogenous interleukin-12 and interferon-gamma production, the source of these Th1-promoting cytokines in infected mice is unknown. In contrast to macrophages from Leishmania-resistant C57BL/6 mice, macrophages from susceptible BALB/c mice are hyporesponsive to stimulation with lipopolysaccharide and interferon-gamma. While studying interactions of various antigen-presenting cells with Leishmania, we found that BALB/c inflammatory skin macrophages, whether Leishmania-infected or uninfected, produced large amounts of interleukin-12 when treated with CpG-containing immunostimulatory oligodeoxynucleotides. Like lipopolysaccharide, CpG-containing immunostimulatory oligodeoxynucleotides induced production of interferon-gamma and release of nitric oxide by skin macrophages. Studies using skin macrophages from interleukin-12- and interferon-gamma-deficient BALB/c mice demonstrated that nitric oxide release was not dependent on interleukin-12 and interferon-gamma production. Approximately 44% and 27% of intracellular Leishmania major amastigotes were killed by infected skin macrophages within 72 h upon stimulation with CpG-containing immunostimulatory oligodeoxynucleotides and lipopolysaccharide, respectively. Parasite killing by macrophages was independent of endogenous interferon-gamma production, but was strongly enhanced by exogenous interferon-gamma. Parasite elimination was dependent on the induction of nitric oxide, however. In vivo, injection of CpG-containing immunostimulatory oligodeoxynucleotides into lesional skin reduced the parasite burden approximately 50-fold within the first 5 d of infection prior to full generation of a Th response. These results suggest that skin macrophages, constituting the principal reservoir of parasites in infected susceptible mice, produce Th1-promoting cytokines in response to CpG-containing immunostimulatory oligodeoxynucleotides. In addition, CpG-containing immunostimulatory oligodeoxynucleotides may also act locally on skin macrophages to facilitate Leishmania clearance by inducing nitric oxide production.

Animals↗
PubMedDate not supplied

Immunological and pathological evaluation of rhesus macaques infected with Leishmania major.

Cutaneous leishmaniasis, a parasitic infection causing ulcerating skin lesions, is an important disease worldwide and urgently requires a vaccine. Animal models that closely mimic human disease are essential for designing preventive vaccines against Leishmania major. We have evaluated both biologic and immunologic parameters of cutaneous L. major infection in nonhuman primates. Naïve rhesus macaques or monkeys previously exposed to L. major were infected with varying doses of L. major metacyclic promastigotes, and lesion size was assessed over a 10-week period. Monkeys previously infected with L. major had much smaller lesions that resolved faster compared with those of naïve monkeys in response to the two higher doses of infection. Moreover, eight of nine naïve monkeys had parasites detected in their lesions during the course of the infection. In addition, the cellular infiltrate within the lesions was qualitatively and quantitatively different in naïve versus previously infected monkeys. Finally, an ELIspot assay determined that the magnitude and kinetics of responses differed between previously infected and naïve monkeys.

Animals↗