Vaccines targeting Staphylococcus aureus skin and bloodstream infections require different composition.

Staphylococcus aureus infections represent a major public health threat, but previous attempts at developing a universal vaccine have been unsuccessful. We attempted to identify a vaccine that would be protective against both skin/soft tissue and bloodstream infections. We first tested a panel of staphylococcal antigens that are conserved across strains, combined with aluminum hydroxide as an adjuvant, for their ability to induce protective immunity in both skin and bacteremia infection models. Antigens were identified that reduced dermonecrosis during skin infection, and other non-overlapping antigens were identified that showed trends to protection in the bacteremia model. However, individual antigens were not identified that mediated substantial protection in both the skin and bacteremia infection models. We therefore tested a variety of combinations of proteins to seek a single combination that could mediate protection in both models. After iterative testing, a vaccine consisting of 3 antigens, ABC transporter protein (SACOL2451), ABC2 transporter protein (SACOL0695), and α-hemolysin (SACOL1173), was identified as the most effective combination. This combination vaccine provided protection in a skin infection model. However, these antigens were only partially protective in the bacteremia infection model. Even by testing multiple different adjuvants, optimized efficacy in the skin infection model did not translate into efficacy in the bacteremia model. Thus protective vaccines against skin/soft tissue infections may not enable effective protection against bloodstream infections.

Monoclonal Antibody Protects Against Acinetobacter baumannii Infection by Enhancing Bacterial Clearance and Evading Sepsis.

Background: Extremely drug-resistant (XDR) Acinetobacter baumannii is one of the most commonly encountered, highly resistant pathogens requiring novel therapeutic interventions. Methods: We developed C8, a monoclonal antibody (mAb), by immunizing mice with sublethal inocula of a hypervirulent XDR clinical isolate. Results: C8 targets capsular carbohydrate on the bacterial surface, enhancing opsonophagocytosis. Treating with a single dose of C8 as low as 0.5 µg/mouse (0.0167 mg/kg) markedly improved survival in lethal bacteremic sepsis and aspiration pneumonia models of XDR A. baumannii infection. C8 was also synergistic with colistin, substantially improving survival compared to monotherapy. Treatment with C8 significantly reduced blood bacterial density, cytokine production (tumor necrosis factor α, interleukin [IL] 6, IL-1ß, and IL-10), and sepsis biomarkers. Serial in vitro passaging of A. baumannii in the presence of C8 did not cause loss of mAb binding to the bacteria, but did result in emergence of less-virulent mutants that were more susceptible to macrophage uptake. Finally, we developed a highly humanized variant of C8 that retains opsonophagocytic activity in murine and human macrophages and rescued mice from lethal infection. Conclusions: We describe a promising and novel mAb as therapy for lethal, XDR A. baumannii infections, and demonstrate that it synergistically improves outcomes in combination with antibiotics.

Diabetes Exacerbates Infection via Hyperinflammation by Signaling through TLR4 and RAGE.

For more than a century, diabetic patients have been considered immunosuppressed due to defects in phagocytosis and microbial killing. We confirmed that diabetic mice were hypersusceptible to bacteremia caused by Gram-negative bacteria (GNB), dying at inocula nonlethal to nondiabetic mice. Contrary to the pervasive paradigm that diabetes impedes phagocytic function, the bacterial burden was no greater in diabetic mice despite excess mortality. However, diabetic mice did exhibit dramatically increased levels of proinflammatory cytokines in response to GNB infections, and immunosuppressing these cytokines with dexamethasone restored their resistance to infection, both of which are consistent with excess inflammation. Furthermore, disruption of the receptor for advanced glycation end products (RAGE), which is stimulated by heightened levels of AGEs in diabetic hosts, protected diabetic but not nondiabetic mice from GNB infection. Thus, rather than immunosuppression, diabetes drives lethal hyperinflammation in response to GNB by signaling through RAGE. As such, interventions to improve the outcomes from GNB infections should seek to suppress the immune response in diabetic hosts.IMPORTANCE Physicians and scientists have subscribed to the dogma that diabetes predisposes the host to worse outcomes from infections because it suppresses the immune system. This understanding was based largely on ex vivo studies of blood from patients and animals with diabetes. However, we have found that the opposite is true and worse outcomes from infection are caused by overstimulation of the immune system in response to bacteria. This overreaction occurs by simultaneous ligation of two host receptors: TLR4 and RAGE. Both signal via a common downstream messenger, MyD88, triggering hyperinflammation. In summary, contrary to hundred-year-old postulations about immune suppression in diabetic hosts, we find that diabetes instead predisposes to more severe infections because of additional inflammatory output through dual activation of MyD88 by not only TLR4 but also RAGE. It is the activation of RAGE during GNB infections in those with diabetes that accounts for their heightened susceptibility to infection compared to nondiabetic hosts.

Long-term exposure of MCF-7 breast cancer cells to ethanol stimulates oncogenic features.

Alcohol consumption is a risk factor for breast cancer. Little is known regarding the mechanism, although it is assumed that acetaldehyde or estrogen mediated pathways play a role. We previously showed that long-term exposure to 2.5 mM ethanol (blood alcohol ~0.012%) of MCF-12A, a human normal epithelial breast cell line, induced epithelial mesenchymal transition (EMT) and oncogenic transformation. In this study, we investigated in the human breast cancer cell line MCF-7, whether a similar exposure to ethanol at concentrations ranging up to peak blood levels in heavy drinkers would increase malignant progression. Short-term (1-week) incubation to ethanol at as low as 1-5 mM (corresponding to blood alcohol concentration of ~0.0048-0.024%) upregulated the stem cell related proteins Oct4 and Nanog, but they were reduced after exposure at 25 mM. Long-term (4-week) exposure to 25 mM ethanol upregulated the Oct4 and Nanog proteins, as well as the malignancy marker Ceacam6. DNA microarray analysis in cells exposed for 1 week showed upregulated expression of metallothionein genes, particularly MT1X. Long-term exposure upregulated expression of some malignancy related genes (STEAP4, SERPINA3, SAMD9, GDF15, KRT15, ITGB6, TP63, and PGR, as well as the CEACAM, interferon related, and HLA gene families). Some of these findings were validated by RT-PCR. A similar treatment also modulated numerous microRNAs (miRs) including one regulator of Oct4 as well as miRs involved in oncogenesis and/or malignancy, with only a few estrogen-induced miRs. Long-term 25 mM ethanol also induced a 5.6-fold upregulation of anchorage-independent growth, an indicator of malignant-like features. Exposure to acetaldehyde resulted in little or no effect comparable to that of ethanol. The previously shown alcohol induction of oncogenic transformation of normal breast cells is now complemented by the current results suggesting alcohol's potential involvement in malignant progression of breast cancer.

Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features.

Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0-2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells.

Ly6G-mediated depletion of neutrophils is dependent on macrophages.

Antibody-mediated depletion of neutrophils is commonly used to study neutropenia. However, the mechanisms by which antibodies deplete neutrophils have not been well defined. We noticed that mice deficient in complement and macrophages had blunted neutrophil depletion in response to anti-Ly6G monoclonal antibody (MAb) treatment. In vitro, exposure of murine neutrophils to anti-Ly6G MAb in the presence of plasma did not result in significant depletion of cells, either in the presence or absence of complement. In vivo, anti-Ly6G-mediated neutrophil depletion was abrogated following macrophage depletion, but not complement depletion, indicating a requirement for macrophages to induce neutropenia by this method. These results inform the use and limitations of anti-Ly6G antibody as an experimental tool for depleting neutrophils in various immunological settings.

Cryopreservation of virulent Acinetobacter baumannii to reduce variability of in vivo studies.

BACKGROUND: Microbiological assays require accurate and reproducible preparation of bacterial inocula. Inocula prepared on different days by different individuals can vary significantly from experiment to experiment. This variance is particularly problematic for Gram-negative bacterial infections, for which threshold effects can result in marked variations in host outcome with minor differences in inocula. RESULTS: We compared the accuracy of traditional methods versus using frozen stocks for preparing Acinetobacter baumannii inocula for infection in mice. Standard inoculum preparation resulted in substantial variability, both with respect to the actual inocula achieved compared to the targeted inocula, and with respect to the in vivo outcome resulting from similar inocula. Cryopreservation of the bacteria resulted in no significant decrement in growth of the bacteria. Furthermore, preparation of multiple infectious inocula from a frozen stock significantly improved the accuracy of the achieved inocula, and resulted in more reproducible in vivo outcomes from infection. Frozen stocks reduced inter-experiment variability associated with inoculum preparation, displayed no significant loss of growth capacity, and maintained virulence, increasing the reliability of infection. CONCLUSIONS: Frozen stocks require considerably less time to prepare and enhance reproducibility of in vivo experimental results when infecting with A. baumannii.

The Potent Humanin Analogue (HNG) Protects Germ Cells and Leucocytes While Enhancing Chemotherapy-Induced Suppression of Cancer Metastases in Male Mice.

Humanin is a peptide that is cytoprotective against stresses in many cell types. We investigated whether a potent humanin analogue S14G-humanin (HNG) would protect against chemotherapy-induced damage to normal cells without interfering with the chemotherapy-induced suppression of cancer cells. Young adult male mice were inoculated iv with murine melanoma cells. After 1 week, cancer-bearing mice were randomized to receive either: no treatment, daily ip injection of HNG, a single ip injection of cyclophosphamide (CP), or CP+HNG and killed at the end of 3 weeks. HNG rescued the CP-induced suppression of leucocytes and protected germ cell from CP-induced apoptosis. Lung metastases were suppressed by HNG or CP alone, and further suppressed by CP+HNG treatment. Plasma IGF-1 levels were suppressed by HNG with or without CP treatment. To investigate whether HNG maintains its protective effects on spermatogonial stem cells, sperm output, and peripheral leucocytes after repeated doses of CP, normal adult male mice received: no treatment, daily sc injection of HNG, 6 ip injections of CP at 5-day intervals, and the same regimens of CP+HNG and killed at the end of 4 weeks of treatment. Cauda epididymal sperm counts were elevated by HNG and suppressed by CP. HNG rescued the CP-induced suppression of spermatogonial stem cells, sperm count and peripheral leucocytes. We conclude that HNG 1) protects CP-induced loss of male germ cells and leucocytes, 2) enhances CP-induced suppression of cancer metastases, and 3) acts as a caloric-restriction mimetic by suppressing IGF-1 levels. Our findings suggest that humanin analogues may be promising adjuvants to chemotherapy.

Transferrin-mediated iron sequestration as a novel therapy for bacterial and fungal infections.

Pathogenic microbes must acquire essential nutrients, including iron, from the host in order to proliferate and cause infections. Iron sequestration is an ancient host antimicrobial strategy. Thus, enhancing iron sequestration is a promising, novel anti-infective strategy. Unfortunately, small molecule iron chelators have proven difficult to develop as anti-infective treatments, in part due to unacceptable toxicities. Iron sequestration in mammals is predominantly mediated by the transferrin family of iron-binding proteins. In this review, we explore the possibility of administering supraphysiological levels of exogenous transferrin as an iron sequestering therapy for infections, which could overcome some of the problems associated with small molecule chelation. Recent studies suggest that transferrin delivery may represent a promising approach to augment both natural resistance and traditional antibiotic therapy.

Host fate is rapidly determined by innate effector-microbial interactions during Acinetobacter baumannii bacteremia.

BACKGROUND: Acinetobacter baumannii is one of the most antibiotic-resistant pathogens. Defining mechanisms driving pathogenesis is critical to enable new therapeutic approaches. METHODS: We studied virulence differences across a diverse panel of A. baumannii clinical isolates during murine bacteremia to elucidate host-microbe interactions that drive outcome. RESULTS: We identified hypervirulent strains that were lethal at low intravenous inocula and achieved very high early, and persistent, blood bacterial densities. Virulent strains were nonlethal at low inocula but lethal at 2.5-fold higher inocula. Finally, relatively avirulent (hypovirulent) strains were nonlethal at 20-fold higher inocula and were efficiently cleared by early time points. In vivo virulence correlated with in vitro resistance to complement and macrophage uptake. Depletion of complement, macrophages, and neutrophils each independently increased bacterial density of the hypovirulent strain but insufficiently to change lethality. However, disruption of all 3 effector mechanisms enabled early bacterial densities similar to hypervirulent strains, rendering infection 100% fatal. CONCLUSIONS: The lethality of A. baumannii strains depends on distinct stages. Strains resistant to early innate effectors are able to establish very high early bacterial blood density, and subsequent sustained bacteremia leads to Toll-like receptor 4-mediated hyperinflammation and lethality. These results have important implications for translational efforts to develop therapies that modulate host-microbe interactions.

Topical resiquimod protects against visceral infection with Leishmania infantum chagasi in mice.

New prevention and treatment strategies are needed for visceral leishmaniasis, particularly ones that can be deployed simply and inexpensively in areas where leishmaniasis is endemic. Synthetic molecules that activate Toll-like receptor 7 and 8 (TLR7/8) pathways have previously been demonstrated to enhance protection against cutaneous leishmaniasis. We initially sought to determine whether the TLR7/8-activating molecule resiquimod might serve as an effective vaccine adjuvant targeting visceral leishmaniasis caused by infection with Leishmania infantum chagasi. Resiquimod was topically applied to the skin of mice either prior to or after systemic infection with L. infantum chagasi, and parasite burdens were assessed. Surprisingly, topical resiquimod application alone, in the absence of vaccination, conferred robust resistance to mice against future intravenous challenge with virulent L. infantum chagasi. This protection against L. infantum chagasi infection persisted as long as 8 weeks after the final topical resiquimod treatment. In addition, in mice with existing infections, therapeutic treatment with topical resiquimod led to significantly lower visceral parasite loads. Resiquimod increased trafficking of leukocytes, including B cells, CD4(+) and CD8(+) T cells, dendritic cells, macrophages, and granulocytes, in livers and spleens, which are the key target organs of visceralizing infection. We conclude that topical resiquimod leads to systemic immune modulation and confers durable protection against visceralizing L. infantum chagasi infection, in both prophylactic and therapeutic settings. These studies support continued studies of TLR-modulating agents to determine mechanisms of protection and also provide a rationale for translational development of a critically needed, novel class of topical, preventative, and therapeutic agents for these lethal infections.

Transferrin iron starvation therapy for lethal bacterial and fungal infections.

New strategies to treat antibiotic-resistant infections are urgently needed. We serendipitously discovered that stem cell conditioned media possessed broad antimicrobial properties. Biochemical, functional, and genetic assays confirmed that the antimicrobial effect was mediated by supra-physiological concentrations of transferrin. Human transferrin inhibited growth of gram-positive (Staphylococcus aureus), gram-negative (Acinetobacter baumannii), and fungal (Candida albicans) pathogens by sequestering iron and disrupting membrane potential. Serial passage in subtherapeutic transferrin concentrations resulted in no emergence of resistance. Infected mice treated with intravenous human transferrin had improved survival and reduced microbial burden. Finally, adjunctive transferrin reduced the emergence of rifampin-resistant mutants of S. aureus in infected mice treated with rifampin. Transferrin is a promising, novel antimicrobial agent that merits clinical investigation. These results provide proof of principle that bacterial infections can be treated in vivo by attacking host targets (ie, trace metal availability) rather than microbial targets.

Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle.

INTRODUCTION: Stimulating the commitment of implanted dystrophin+ muscle-derived stem cells (MDSCs) into myogenic, as opposed to lipofibrogenic lineages, is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). METHODS: To examine whether counteracting myostatin, a negative regulator of muscle mass and a pro-lipofibrotic factor, would help this process, we compared the in vitro myogenic and fibrogenic capacity of MDSCs from wild-type (WT) and myostatin knockout (Mst KO) mice under various modulators, the expression of key stem cell and myogenic genes, and the capacity of these MDSCs to repair the injured gastrocnemius in aged dystrophic mdx mice with exacerbated lipofibrosis. RESULTS: Surprisingly, the potent in vitro myotube formation by WT MDSCs was refractory to modulators of myostatin expression or activity, and the Mst KO MDSCs failed to form myotubes under various conditions, despite both MDSC expressing Oct 4 and various stem cell genes and differentiating into nonmyogenic lineages. The genetic inactivation of myostatin in MDSCs was associated with silencing of critical genes for early myogenesis (Actc1, Acta1, and MyoD). WT MDSCs implanted into the injured gastrocnemius of aged mdx mice significantly improved myofiber repair and reduced fat deposition and, to a lesser extent, fibrosis. In contrast to their in vitro behavior, Mst KO MDSCs in vivo also significantly improved myofiber repair, but had few effects on lipofibrotic degeneration. CONCLUSIONS: Although WT MDSCs are very myogenic in culture and stimulate muscle repair after injury in the aged mdx mouse, myostatin genetic inactivation blocks myotube formation in vitro, but the myogenic capacity is recovered in vivo under the influence of the myostatin+ host-tissue environment, presumably by reactivation of key genes originally silenced in the Mst KO MDSCs.

Oxidized low-density lipoprotein antigen transport induces autoimmunity in the renal tubulointerstitium.

BACKGROUND/AIMS: Chronic kidney disease involves inflammation/oxidative stress, which contributes to progressive kidney injury. METHODS: Male Sprague-Dawley rats underwent 5/6 nephrectomy (Nx) or sham Nx and were sacrificed after 2 days, 2 weeks and 4 weeks. Microarray analysis expression sets over time suggested the evolution of renal lymphocyte infiltration and antigen-presenting cell (APC) activation after 5/6Nx. RT-PCR analysis also confirmed the migration and activation of lymphocytes and APCs through the upregulation of CD3, CXCR3/CXCL10 and CCR7/CCL19 mRNA in remnant kidney (RK). Purified T lymphocytes from spleen and unilateral ureteral obstruction (UUO) kidney were incubated with oxidized low-density lipoprotein (Ox-LDL)-treated major histocompatibility complex class II (MHC II)-expressing APCs. Culture supernatant was collected for mouse IFN-γ ELISA and cell proliferation was measured. RESULTS: Ox-LDL deposited predominantly in renal tubulointerstitial areas of RK, increased over time, and co-stained with lectin-like Ox-LDL receptor in affected renal tubular cells. Both Ox-LDL and renal-specific glycoprotein Tamm-Horsfall protein were identified in renal lymph nodes. Cells co-staining for major MHC II and Ox-LDL were observed in RK and draining renal lymph nodes after 5/6Nx. Similarly, Ox-LDL was also present in tubules after UUO, CD3-positive T cells were present in the interstitium, and Ox-LDL-treated MHC II-expressing APCs induced proliferation and IFN-γ production in renal tubulointerstitial T lymphocytes isolated from kidneys after UUO. CONCLUSIONS: These data demonstrate that the tubulointerstitial inflammatory infiltrate that accompanies chronic kidney disease reflects, at least in part, the development of autoimmunity to novel antigens generated during renal injury.

Killed but metabolically active Leishmania infantum as a novel whole-cell vaccine for visceral leishmaniasis.

There are currently no effective vaccines for visceral leishmaniasis, the second most deadly parasitic infection in the world. Here, we describe a novel whole-cell vaccine approach using Leishmania infantum chagasi promastigotes treated with the psoralen compound amotosalen (S-59) and low doses of UV A radiation. This treatment generates permanent, covalent DNA cross-links within parasites and results in Leishmania organisms termed killed but metabolically active (KBMA). In this report, we characterize the in vitro growth characteristics of both KBMA L. major and KBMA L. infantum chagasi. Concentrations of S-59 that generate optimally attenuated parasites were identified. Like live L. infantum chagasi, KBMA L. infantum chagasi parasites were able to initially enter liver cells in vivo after intravenous infection. However, whereas live L. infantum chagasi infection leads to hepatosplenomegaly in mice after 6 months, KBMA L. infantum chagasi parasites were undetectable in the organs of mice at this time point. In vitro, KBMA L. infantum chagasi retained the ability to enter macrophages and induce nitric oxide production. These characteristics of KBMA L. infantum chagasi correlated with the ability to prophylactically protect mice via subcutaneous vaccination at levels similar to vaccination with live, virulent organisms. Splenocytes from mice vaccinated with either live L. infantum chagasi or KBMA L. infantum chagasi displayed similar cytokine patterns in vitro. These results suggest that KBMA technology is a potentially safe and effective novel vaccine strategy against the intracellular protozoan L. infantum chagasi. This approach may represent a new method for whole-cell vaccination against other complex intracellular pathogens.

Delineation of antigen-specific and antigen-nonspecific CD8(+) memory T-cell responses after cytokine-based cancer immunotherapy.

Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8(+) T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8(+) T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8(+) T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8(+) CD25(-) cells within the tumor site. These findings demonstrate that memory CD8(+) T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.

Efficacy of synthetic peptides RP-1 and AA-RP-1 against Leishmania species in vitro and in vivo.

Host defense peptides are naturally occurring molecules that play essential roles in innate immunity to infection. Based on prior structure-function knowledge, we tested two synthetic peptides (RP-1 and AA-RP-1) modeled on the conserved, microbicidal α-helical domain of mammalian CXCL4 platelet kinocidins. These peptides were evaluated for efficacy against Leishmania species, the causative agents of the group of diseases known as leishmaniasis. In vitro antileishmanial activity was assessed against three distinct Leishmania strains by measuring proliferation, metabolic activity and parasite viability after exposure to various concentrations of peptides. We demonstrate that micromolar concentrations of RP-1 and AA-RP-1 caused dose-dependent growth inhibition of Leishmania promastigotes. This antileishmanial activity correlated with rapid membrane disruption, as well as with a loss of mitochondrial transmembrane potential. In addition, RP-1 and AA-RP-1 demonstrated distinct and significant in vivo antileishmanial activities in a mouse model of experimental visceral leishmaniasis after intravenous administration. These results establish efficacy of RP-1 lineage synthetic peptides against Leishmania species in vitro and after intravenous administration in vivo and provide further validation of proof of concept for the development of these and related systemic anti-infective peptides targeting pathogens that are resistant to conventional antibiotics.

Immunomodulation by imiquimod in patients with high-risk primary melanoma.

Imiquimod is a synthetic Toll-like receptor 7 (TLR7) agonist approved for the topical treatment of actinic keratoses, superficial basal cell carcinoma, and genital warts. Imiquimod leads to an 80-100% cure rate of lentigo maligna; however, studies of invasive melanoma are lacking. We conducted a pilot study to characterize the local, regional, and systemic immune responses induced by imiquimod in patients with high-risk melanoma. After treatment of the primary melanoma biopsy site with placebo or imiquimod cream, we measured immune responses in the treated skin, sentinel lymph nodes (SLNs), and peripheral blood. Treatment of primary melanomas with 5% imiquimod cream was associated with an increase in both CD4+ and CD8+ T cells in the skin, and CD4+ T cells in the SLN. Most of the CD8+ T cells in the skin were CD25 negative. We could not detect any increases in CD8+ T cells specifically recognizing HLA-A(*)0201-restricted melanoma epitopes in the peripheral blood. The findings from this small pilot study demonstrate that topical imiquimod treatment results in enhanced local and regional T-cell numbers in both the skin and SLN. Further research into TLR7 immunomodulating pathways as a basis for effective immunotherapy against melanoma in conjunction with surgery is warranted.

A critical role for GRP78/BiP in the tumor microenvironment for neovascularization during tumor growth and metastasis.

Glucose-regulated protein 78 (GRP78)/BiP is a multifunctional protein which plays a major role in endoplasmic reticulum (ER) protein processing, protein quality control, maintaining ER homeostasis, and controlling cell signaling and viability. Previously, using a transgene-induced mammary tumor model, we showed that Grp78 heterozygosity impeded cancer growth through suppression of tumor cell proliferation and promotion of apoptosis and the Grp78(+/-) mice exhibited dramatic reduction (70%) in the microvessel density (MVD) of the endogenous mammary tumors, while having no effect on the MVD of normal organs. This observation suggests that GRP78 may critically regulate the function of the host vasculature within the tumor microenvironment. In this article, we interrogated the role of GRP78 in the tumor microenvironment. In mouse tumor models in which wild-type (WT), syngeneic mammary tumor cells were injected into the host, we showed that Grp78(+/-) mice suppressed tumor growth and angiogenesis during the early phase but not during the late phase of tumor growth. Growth of metastatic lesions of WT, syngeneic melanoma cells in the Grp78(+/-) mice was potently suppressed. We created conditional heterozygous knockout of GRP78 in the host endothelial cells and showed severe reduction of tumor angiogenesis and metastatic growth, with minimal effect on normal tissue MVD. Furthermore, knockdown of GRP78 expression in immortalized human endothelial cells showed that GRP78 is a critical mediator of angiogenesis by regulating cell proliferation, survival, and migration. Our findings suggest that concomitant use of current chemotherapeutic agents and novel therapies against GRP78 may offer a powerful dual approach to arrest cancer initiation, progression, and metastasis.