A First Phenotypic and Functional Characterization of Placental Extracellular Vesicles from Women with Multiple Sclerosis.

Pregnancy is a unique situation of physiological immunomodulation, as well as a strong Multiple Sclerosis (MS) disease modulator whose mechanisms are still unclear. Both maternal (decidua) and fetal (trophoblast) placental cells secrete extracellular vesicles (EVs), which are known to mediate cellular communication and modulate the maternal immune response. Their contribution to the MS disease course during pregnancy, however, is unexplored. Here, we provide a first phenotypic and functional characterization of EVs isolated from cultures of term placenta samples of women with MS, differentiating between decidua and trophoblast. In particular, we analyzed the expression profile of 37 surface proteins and tested the functional role of placental EVs on mono-cultures of CD14+ monocytes and co-cultures of CD4+ T and regulatory T (Treg) cells. Results indicated that placental EVs are enriched for surface markers typical of stem/progenitor cells, and that conditioning with EVs from samples of women with MS is associated to a moderate decrease in the expression of proinflammatory cytokines by activated monocytes and in the proliferation rate of activated T cells co-cultured with Tregs. Overall, our findings suggest an immunomodulatory potential of placental EVs from women with MS and set the stage for a promising research field aiming at elucidating their role in MS remission.

TNFAIP3 Deficiency Affects Monocytes, Monocytes-Derived Cells and Microglia in Mice.

The intracellular-ubiquitin-ending-enzyme tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is a potent inhibitor of the pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) pathway. Single nucleotide polymorphisms in TNFAIP3 locus have been associated to autoimmune inflammatory disorders, including Multiple Sclerosis (MS). Previously, we reported a TNFAIP3 down-regulated gene expression level in blood and specifically in monocytes obtained from treatment-naïve MS patients compared to healthy controls (HC). Myeloid cells exert a key role in the pathogenesis of MS. Here we evaluated the effect of specific TNFAIP3 deficiency in myeloid cells including monocytes, monocyte-derived cells (M-MDC) and microglia analyzing lymphoid organs and microglia of mice. TNFAIP3 deletion is induced using conditional knock-out mice for myeloid lineage. Flow-cytometry and histological procedures were applied to assess the immune cell populations of spleen, lymph nodes and bone marrow and microglial cell density in the central nervous system (CNS), respectively. We found that TNFAIP3 deletion in myeloid cells induces a reduction in body weight, a decrease in the number of M-MDC and of common monocyte and granulocyte precursor cells (CMGPs). We also reported that the lack of TNFAIP3 in myeloid cells induces an increase in microglial cell density. The results suggest that TNFAIP3 in myeloid cells critically controls the development of M-MDC in lymphoid organ and of microglia in the CNS.

Immunomodulatory Effect of Pregnancy on Leukocyte Populations in Patients With Multiple Sclerosis: A Comparison of Peripheral Blood and Decidual Placental Tissue.

Pregnancy is a naturally occurring disease modifier of multiple sclerosis (MS) associated with a substantial reduction in relapse rate. To date, attempts to explain this phenomenon have focused on systemic maternal immune cell composition, with contradictory results. To address this matter, we compared the immunomodulatory effects of pregnancy on five leukocyte populations (i.e., CD4+ and CD8+ T cells, CD4+CD127-CD25high regulatory T cells, CD56brightCD16- NK cells, and CD14+CD163+ monocytes) in peripheral blood from different cohorts of MS patients and healthy women at different times of gestation, as well as in decidual samples from the placenta of MS patients and healthy women collected after delivery. For the first time to our knowledge, we observed that the frequency of these cell populations in the decidua is not different between MS patients and healthy women, suggesting that a physiological immune regulation may occur at the fetal-maternal interface. In peripheral blood, however, contrary to healthy women, in MS patients cell frequencies were not significantly altered by gestation. In particular, CD8+ T cells did not show differences between groups. CD4+ T cells were higher in non-pregnant MS compared to healthy women, while during pregnancy they remained constant in MS and increased in healthy women. Regulatory T cells were higher in non-pregnant controls compared to MS women, while the difference was reduced during gestation due to the decrease of regulatory T cell levels in healthy women. CD14+CD163+ monocytes did not show differences between groups. CD56brightCD16- NK cells were not significantly different in non-pregnant MS compared to controls and increased in healthy women during gestation. In conclusion, our findings support the hypothesis that disease amelioration in MS patients during pregnancy may be due to a modulation of the immune cells functional activity rather than their frequency. Further studies exploring functional changes of these cells would be crucial to bring light into the complex mechanisms of pregnancy-induced tolerance and autoimmunity overall.

NURR1 deficiency is associated to ADHD-like phenotypes in mice.

The transcription factor NURR1 regulates the dopamine (DA) signaling pathway and exerts a critical role in the development of midbrain dopaminergic neurons (mDA). NURR1 alterations have been linked to DA-associated brain disorders, such as Parkinson's disease and schizophrenia. However, the association between NURR1 defects and the attention-deficit hyperactivity disorder (ADHD), a DA-associated brain disease characterized by hyperactivity, impulsivity and inattention, has never been demonstrated. To date, a comprehensive murine model of ADHD truly reflecting the whole complex human psychiatric disorder still does not exist. NURR1-knockout (NURR1-KO) mice have been reported to exhibit increased spontaneous locomotor activity, but their complete characterization is still lacking. In the present study a wide-ranging test battery was used to perform a comprehensive analysis of the behavioral phenotype of the male NURR1-KO mice. As a result, their hyperactive phenotype was confirmed, while their impulsive behavior was reported for the first time. On the other hand, no anxiety and alterations in motor coordination, sociability and memory were observed. Also, the number of mDA expressing tyrosine hydroxylase, a rate-limiting enzyme of catecholamines biosynthesis, and DA level in brain were not impaired in NURR1-KO mice. Finally, hyperactivity has been shown to be recovered by treatment with methylphenidate, the first line psychostimulant drug used for ADHD. Overall, our study suggests that the NURR1 deficient male mouse may be a satisfactory model to study some ADHD behavioral phenotypes and to test the clinical efficacy of potential therapeutic agents.

The Footprints of Poly-Autoimmunity: Evidence for Common Biological Factors Involved in Multiple Sclerosis and Hashimoto's Thyroiditis.

Autoimmune diseases are a diverse group of chronic disorders and affect a multitude of organs and systems. However, the existence of common pathophysiological mechanisms is hypothesized and reports of shared risk are emerging as well. In this regard, patients with multiple sclerosis (MS) have been shown to have an increased susceptibility to develop chronic autoimmune thyroid diseases, in particular Hashimoto's thyroiditis (HT), suggesting an autoimmune predisposition. However, studies comparing such different pathologies of autoimmune origin are still missing till date. In the present study, we sought to investigate mechanisms which may lead to the frequent coexistence of MS and HT by analyzing several factors related to the pathogenesis of MS and HT in patients affected by one or both diseases, as well as in healthy donors. In particular, we analyzed peripheral blood mononuclear cell gene-expression levels of common candidate genes such as TNFAIP3, NR4A family, BACH2, FOXP3, and PDCD5, in addition to the regulatory T cell (Treg) percentage and the 25-hydroxy vitamin D serum levels. Our findings support the plausibility of the existence of common deregulated mechanisms shared by MS and HT, such as BACH2/PDCD5-FOXP3 pathways and Tregs. Although the biological implications of these data need to be further investigated, we have highlighted the relevance of studies comparing different autoimmune pathologies for the understanding of the core concepts of autoimmunity.

Biological activity of glatiramer acetate on Treg and anti-inflammatory monocytes persists for more than 10years in responder multiple sclerosis patients.

Glatiramer acetate (GA) is a widely used treatment for multiple sclerosis (MS), with incompletely defined mechanism of action. Short-term studies suggested its involvement in the modulation of anti-inflammatory cytokines and regulatory T cells (Treg), while long-term effect is still unknown. To investigate this aspect, we analyzed by flow-cytometry peripheral-blood Treg, natural killer (NK), CD4 and CD8 T-cells and anti-inflammatory CD14+CD163+ monocytes from 37 healthy donor and 90 RRMS patients divided in untreated, treated with GA for 12months and from 34 to 192months. While NK, CD4 and CD8 T-cells did not show any significant differences among groups over time, we demonstrated that GA increased the anti-inflammatory monocytes and restored the Treg level in both GA-treated groups. Both these effects are a characteristic of responder patients and are observed not just in short-term but even after as long as a decade of GA treatment.

PML risk stratification using anti-JCV antibody index and L-selectin.

BACKGROUND: Natalizumab treatment is associated with progressive multifocal leukoencephalopathy (PML) development. Treatment duration, prior immunosuppressant use, and JCV serostatus are currently used for risk stratification, but PML incidence stays high. Anti-JCV antibody index and L-selectin (CD62L) have been proposed as additional risk stratification parameters. OBJECTIVE: This study aimed at verifying and integrating both parameters into one algorithm for risk stratification. METHODS: Multicentric, international cohorts of natalizumab-treated MS patients were assessed for JCV index (1921 control patients and nine pre-PML patients) and CD62L (1410 control patients and 17 pre-PML patients). RESULTS: CD62L values correlate with JCV serostatus, as well as JCV index values. Low CD62L in natalizumab-treated patients was confirmed and validated as a biomarker for PML risk with the risk factor "CD62L low" increasing a patient's relative risk 55-fold (p < 0.0001). Validation efforts established 86% sensitivity/91% specificity for CD62L and 100% sensitivity/59% specificity for JCV index as predictors of PML. Using both parameters identified 1.9% of natalizumab-treated patients in the reference center as the risk group. CONCLUSIONS: Both JCV index and CD62L have merit for risk stratification and share a potential biological relationship with implications for general PML etiology. A risk algorithm incorporating both biomarkers could strongly reduce PML incidence.

Natalizumab treatment reduces L-selectin (CD62L) in CD4+ T cells.

BACKGROUND: The purpose of this research was to validate the low expression of L-selectin (CD62L) in natalizumab (NTZ)-treated patients. CD62L is involved in rolling and transmigration of leukocyte cells. A correlation between CD62LCD4+ T cells low expression and progressive multifocal leukoencephalopathy (PML) development has been suggested in multiple sclerosis (MS) patients treated with NTZ. METHODS: We performed a flow cytometric analysis on peripheral blood mononuclear cells (PBMC); we collected from 23 healthy donors and 225 MS patients: untreated (n = 19) or treated with NTZ (n = 113), interferon-beta (n = 26), glatiramer acetate (n = 26), fingolimod (n = 23) and rituximab (n = 18). We have also analysed two PML/IRIS (immune reconstitution inflammatory syndrome) patients and four longitudinal samples of a NTZ-treated patients before and during the development of a clinical asymptomatic magnetic resonance imaging (MRI) lesion confirmed as PML by cerebrospinal fluid (CSF) examination. Thirty-five NTZ-treated patients were studied longitudinally with three samples taken 4 months apart. RESULTS: The NTZ-treated patients showed a lower percentage of CD62L (33.68%, n = 113) than first-line treated patients (44.24%, n = 52, p = 0.0004). NTZ effect was already clear during the first year of treatment (34.68 ; p = 0.0184); it persisted in the following years and disappeared after drug withdrawal (44.08%). Three percent of longitudinally analysed patients showed a percentage of CD62LCD4+ T cells under a hypothetical threshold and one patient with asymptomatic PML belongs to a group which expressed low percentage of CD62LCD4+ T cells. CONCLUSIONS: Our research confirms that NTZ has a specific effect on CD62LCD4+ T cells consisting in decreasing of the number of positive cells. The low level of CD62L found in a clinically asymptomatic PML patient strengthens its potential usefulness as a biomarker of high PML risk in NTZ-treated patients. A larger study is required to better confirm the data.

Antitumor immunization of mothers delays tumor development in cancer-prone offspring.

Maternal immunization is successfully applied against some life-threatening infectious diseases as it can protect the mother and her offspring through the passive transfer of maternal antibodies. Here, we sought to evaluate whether the concept of maternal immunization could also be applied to cancer immune-prevention. We have previously shown that antibodies induced by DNA vaccination against rat Her2 (neu) protect heterozygous neu-transgenic female (BALB-neuT) mice from autochthonous mammary tumor development. We, herein, seek to evaluate whether a similar maternal immunization can confer antitumor protection to BALB-neuT offspring. Significantly extended tumor-free survival was observed in BALB-neuT offspring born and fed by mothers vaccinated against neu, as compared to controls. Maternally derived anti-neu immunoglobulin G (IgG) was successfully transferred from mothers to newborns and was responsible for the protective effect. Vaccinated mothers and offspring also developed active immunity against neu as revealed by the presence of T-cell-mediated cytotoxicity against the neu immunodominant peptide. This active response was due to the milk transfer of immune complexes that were formed between the neu extracellular domain, shed from vaccine-transfected muscle cells, and the anti-neu IgG induced by the vaccine. These findings show that maternal immunization has the potential to hamper mammary cancer in genetically predestinated offspring and to develop into applications against lethal neonatal cancer diseases for which therapeutic options are currently unavailable.

Recombinant human lactoferrin induces human and mouse dendritic cell maturation via Toll-like receptors 2 and 4.

Lactoferrin, a key component of innate immunity, is a cationic monomeric 80-kDa glycoprotein of the transferrin superfamily. Recombinant human lactoferrin, known as talactoferrin (TLF), induces a distinct functional maturation program in human dendritic cells (DCs) derived from peripheral blood monocytes. However, the receptors and molecular mechanisms involved in this induction have not been fully determined. By exploiting genome-wide transcription profiling of immature DCs, TNF-α- and IL-1ß-matured DCs (m-DCs), and TLF-matured DCs (TLF-DCs), we have detected a set of transcripts specific for m-DCs and one specific for TLF-DCs. Functional network reconstruction highlighted, as expected, the association of m-DC maturation with IL-1ß, TNF-α, and NF-κB, whereas TLF-DC maturation was associated with ERK and NF-κB. This involvement of ERK and NF-κB transduction factors suggests direct involvement of Toll-like receptors (TLRs) in TLF-induced maturation. We have used MyD88 inhibition and siRNA silencing TLRs on human DCs and mouse TLR-2-knockout cells, to show that TLF triggers the maturation of both human and mouse DCs through TLR-2 and TLR-4.

p130Cas alters the differentiation potential of mammary luminal progenitors by deregulating c-Kit activity.

It has recently been proposed that defective differentiation of mammary luminal progenitors predisposes to basal-like breast cancer. However, the molecular and cellular mechanisms involved are still unclear. Here, we describe that the adaptor protein p130Cas is a crucial regulator of mouse mammary epithelial cell (MMEC) differentiation. Using a transgenic mouse model, we show that forced p130Cas overexpression in the luminal progenitor cell compartment results in the expansion of luminal cells, which aberrantly display basal cell features and reduced differentiation in response to lactogenic stimuli. Interestingly, MMECs overexpressing p130Cas exhibit hyperactivation of the tyrosine kinase receptor c-Kit. In addition, we demonstrate that the constitutive c-Kit activation alone mimics p130Cas overexpression, whereas c-Kit downregulation is sufficient to re-establish proper differentiation of p130Cas overexpressing cells. Overall, our data indicate that high levels of p130Cas, via abnormal c-Kit activation, promote mammary luminal cell plasticity, thus providing the conditions for the development of basal-like breast cancer. Consistently, p130Cas is overexpressed in human triple-negative breast cancer, further suggesting that p130Cas upregulation may be a priming event for the onset of basal-like breast cancer.

A DNA vaccine against ERBB2 impairs chemical carcinogenesis in random-bred hamsters.

Vaccines against oncoantigens halt early neoplastic lesions in several cancer-prone, genetically engineered mouse models, whereas their ability to prevent chemical carcinogenesis has not been explored. This is a significant issue, as exposure to chemical mutagens is responsible for a substantial percentage of cancers worldwide. Here, we show that the archetypal oncoantigen ERBB2 is transiently overexpressed in Syrian hamsters during the early stages of 7,12-dimethylbenz[α]anthracene (DMBA)-induced oral carcinogenesis. Repeated DNA vaccinations against ERBB2 significantly reduce the number, size, and severity of oral lesions in a manner directly proportional to the anti-ERBB2 antibody response. These results support the prospects of vaccines as a fresh strategy in the management of individuals at risk for exposure to defined carcinogenic agents.

Erbb2 DNA vaccine combined with regulatory T cell deletion enhances antibody response and reveals latent low-avidity T cells: potential and limits of its therapeutic efficacy.

Rat (r)Erbb2 transgenic BALB-neuT mice genetically predestined to develop multiple invasive carcinomas allow an assessment of the potential of a vaccine against the stages of cancer progression. Because of rErbb2 expression in the thymus and its overexpression in the mammary gland, CD8(+) T cell clones reacting at high avidity with dominant rErbb2 epitopes are deleted in these mice. In BALB-neuT mice with diffuse and invasive in situ lesions and almost palpable carcinomas, a temporary regulatory T cells depletion combined with anti-rErbb2 vaccine markedly enhanced the anti-rErbb2 Ab response and allowed the expansion of latent pools of low-avidity CD8(+) T cells bearing TCRs repertoire reacting with the rErbb2 dominant peptide. This combination of a higher Ab response and activation of a low-avidity cytotoxic response persistently blocked tumor progression at stages in which the vaccine alone was ineffective. However, when diffuse and invasive microscopic cancers become almost palpable, this combination was no longer able to secure a significant extension of mice survival.

In vivo MRI multicontrast kinetic analysis of the uptake and intracellular trafficking of paramagnetically labeled liposomes.

This work aims at developing a MRI method that allows to get more insight into the understanding of the in vivo fate of liposomes and their payload. The method relies on the temporal assessment of the contrast changes induced by the presence of a classical relaxation agent versus the effect induced by a CEST (chemical exchange saturation transfer) agent. Liposomes were loaded with the paramagnetic complexes, Gd-HPDO3A and [Tm-DOTMA](-) [Na](+), in order to endow the nanovesicles with the characteristic properties of T(1)/T(2) and CEST/T(2) MRI agents, respectively. The paramagnetically loaded liposomes were injected directly into the tumor (B16 melanoma xenograft in mice) where they generate T(1), T(2), and CEST MR contrasts that were quantitatively monitored over time (0-48h). The kinetic of each contrast enhancement reports about peculiar properties relative to the fate of the liposomes in the tumor environment. A kinetic model has been set-up to fit the experimental multicontrast data in order to extract the relevant information about the cellular uptake of the liposomes and the release of their payload. Upon comparing conventional stealth liposomes with pH-sensitive ones, it has been shown that the latter ones differ essentially in the step associated with the release of the drug that is likely occurring in the endosomal acidic vesicles.

Lactoferrin, a major defense protein of innate immunity, is a novel maturation factor for human dendritic cells.

Lactoferrin (LF) is an important protein component of the innate immune system that is broadly distributed within the body fluids. LF is endowed with multiple biological activities. Talactoferrin (TLF), a recombinant human LF, is in clinical development as an anticancer agent and is entering Phase III clinical trials. Here, we show that TLF induces the maturation of human dendritic cells (DCs) derived from monocytes. TLF, at physiologically relevant concentrations (100 microg/ml) up-regulates the expression of human leukocyte antigen (HLA) class II, CD83, CD80, and CD86 costimulatory molecule and CXCR4 and CCR7 chemokine receptors, acting primarily through the p38 MAPK signaling pathway. DCs matured by TLF displayed an enhanced release of IL-8 and CXCL10, as well as a significantly reduced production of IL-6, IL-10, and CCL20. They also display a reduced ability to take up antigen and increased capacity to trigger proliferation and release IFN-gamma in the presence of allogeneic human T cells. TLF-matured DCs are able to prime naive T cells to respond to KLH antigen and display a significantly increased capacity to present Flu-MA(58-66) peptide to HLA-A2-matched T cells. These data suggest that a key immunomodulatory function that may be mediated by TLF is to link the innate with adaptive immunity through DC maturation.

Requirement for IFN-gamma, CD8+ T lymphocytes, and NKT cells in talactoferrin-induced inhibition of neu+ tumors.

We have previously shown that talactoferrin-alfa (TLF), a recombinant human lactoferrin, is an immunomodulatory protein that is active against implanted tumors, both as a single agent and in combination with chemotherapy. In this study, we show that talactoferrin is active against autochthonous tumors in a transgenic mouse line, which is more analogous to human cancers, and identify key mechanistic steps involved in the anticancer activity of oral TLF. BALB/c mice transgenic for the rat neu (ErbB2) oncogene (BALB-neuT) treated with oral TLF showed a significant delay in carcinogenesis, with 60% tumor protection relative to vehicle-treated mice at week 21. Oral TLF also showed tumor growth inhibition in wild-type BALB/c mice implanted with neu(+) mammary adenocarcinoma, with one third displaying a long-lasting or complete response. Oral TLF induces an increase in intestinal mucosal IFN-gamma production and an increase in Peyer's patch cellularity, including expansion of CD8(+) T lymphocytes and NKT cells, and the enhancement of CD8(+) T-cell cytotoxicity. In IFN-gamma knockout mice, there is an absence of the TLF-induced Peyer's patch cellularity, no expansion of CD8(+) T lymphocytes and NKT cells, and loss of TLF anticancer activity. TLF antitumor activity is also lost in mice depleted of CD8(+) T cells and in CD1 knockout mice, which lack NKT activity. Thus, the inhibition of distant tumors by oral TLF seems to be mediated by an IFN-gamma-dependent enhancement of CD8(+) T- and NKT cell activity initiated within the intestinal mucosa.

Immunosurveillance of Erbb2 carcinogenesis in transgenic mice is concealed by a dominant regulatory T-cell self-tolerance.

To assess the role of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells in overcoming immunosurveillance of Erbb2 (HER-2/neu) mammary lesions, we studied the effects of their sustained removal in BALB/c female mice made transgenic for the rat Erbb2 (r-Erbb2) oncogene (BALB-neuT mice), which develop multiple mammary carcinomas. During the progression of these lesions, Treg cells expand in the spleen, tumor draining lymph nodes, and tumors. Repeated administration of anti-CD25 antibodies extends tumor-free survival, reduces carcinoma multiplicity, and leads to the manifestation of a natural antibody and CTL-mediated reactivity against r-Erbb2. Loss of Foxp3(+) Treg cells during anti-CD25 treatment remarkably caused the disappearance of Gr1(+) immature myeloid cells, suggesting a cross-talk between these two inhibitory immune cell types. Treg cell expansion associated with r-Erbb2 overexpression may be seen as a physiologic response to dampen the immune reaction elicited by local anomalous overexpression of a self-antigen.

Timely DNA vaccine combined with systemic IL-12 prevents parotid carcinomas before a dominant-negative p53 makes their growth independent of HER-2/neu expression.

Double transgenic mice overexpressing the transforming rat HER-2/neu oncogene and the mutated p53, with both dominant-negative and a gain-of-function properties, display early aggressive and metastasizing parotid tumors. Multiple acinar and ductal hyperplasia foci overexpressing the HER-2/neu gene product are evident at wk 5 and progress to poorly differentiated carcinoma by wk 7. Mice die before wk 18 with invasive carcinomas and multiple metastases that no longer express HER-2/neu. A combination of repeated electroporations of plasmids coding for the extracellular and transmembrane domains of the rat HER-2/neu receptor with systemic IL-12 administrations started when the parotids that present diffuse hyperplasia protected all female and 50% of the male mice until the close of the experiment at wk 40. This combined treatment began when multifocal in situ carcinomas that were already present cured 33% of the females and 25% of the males. The most prominent immunologic features associated with the antitumor protection were the production of high titers of anti-HER-2/neu Abs and the nonappearance of cell-mediated cytotoxic reactivity. In conclusion, anti-HER-2/neu vaccination combined with systemic IL-12 control parotid carcinomas as far as p53 mutation makes their growth independent of HER-2/neu expression.

Cure of mammary carcinomas in Her-2 transgenic mice through sequential stimulation of innate (neoadjuvant interleukin-12) and adaptive (DNA vaccine electroporation) immunity.

PURPOSE: Whereas neoadjuvant therapy is emerging as a treatment option in early primary breast cancer, no data are available on the use of antiangiogenic and immunomodulatory agents in a neoadjuvant setting. In a model of Her-2 spontaneous mammary cancer, we investigated the efficacy of neoadjuvant interleukin 12 (IL-12) followed by "immune-surgery" of the residual tumor. EXPERIMENTAL DESIGN: Female BALB/c mice transgenic for the rat Her-2 oncogene inexorably develop invasive carcinomas in all their mammary glands by the 23rd week of age. Mice with multifocal in situ carcinomas received four weekly i.p. injections of 100 ng IL-12 followed by a 3-week rest. This course was given four times. A few mice additionally received DNA plasmids encoding portions of the Her-2 receptor electroporated through transcutaneous electric pulses. RESULTS: The protection elicited by IL-12 in combination with two DNA vaccine electroporations kept 63% of mice tumor-free. Complete protection of all 1-year-old mice was achieved when IL-12-treated mice received four vaccine electroporations. Pathologic findings, in vitro tests, and the results from immunization of both IFN-gamma and immunoglobulin gene knockout transgenic mice and of adoptive transfer experiments all show that IL-12 augments the B- and T-cell response elicited by vaccination and slightly decreases the number of regulatory T cells. In addition, IL-12 strongly inhibits tumor angiogenesis. CONCLUSIONS: In Her-2 transgenic mice, IL-12 impairs tumor progression and triggers innate immunity so markedly that DNA vaccination becomes effective at late points in time when it is ineffective on its own.

Oral lactoferrin inhibits growth of established tumors and potentiates conventional chemotherapy.

In this work, we investigated the anticancer activity of orally administered recombinant human lactoferrin (rhLF) alone and in combination with chemotherapy in tumor-bearing mice. rhLF inhibited the growth of squamous cell carcinoma (O12) tumors in T cell-immunocompromised nu/nu mice by 80% when administered at 1,000 mg/kg (2.9 g/m2) by oral gavage twice daily for 8 days (p < 0.001). Similar activity was observed in syngeneic, immunocompetent BALB/c mice, where orally administered rhLF (1,000 mg/kg, 2.9 g/m2 once daily) halted the growth of mammary adenocarcinoma TUBO. Oral rhLF (200 mg/kg, 0.57 g/m2) was also used alone and in combination with cis-platinum (5 mg/kg) to treat head-and-neck squamous cell carcinoma in a syngeneic murine model. Monotherapy with oral rhLF or cis-platinum caused 61% or 66% tumor growth inhibition over placebo, respectively. Mice receiving both therapies showed 79% growth inhibition, a statistically significant improvement over each drug alone. We then demonstrated that administration of oral rhLF (300 mg/kg, 0.86 g/m2) to tumor-bearing or naive mice resulted in (i) significantly increased production of IL-18 in the intestinal tract, (ii) systemic NK cell activation and (iii) circulating CD8+ T-cell expansion. These data suggest that oral rhLF is an immunomodulatory agent active against cancer as a single agent and in combination chemotherapy, exerting its systemic effect through stimulation of IL-18 and other cytokines in the gut enterocytes. rhLF has been administered orally to 211 people without a single serious drug-related adverse event. Thus, rhLF shows promise as a safe and well-tolerated novel immunomodulatory anticancer agent.