Macrophage migration inhibitory factor in Nodding syndrome.

Nodding syndrome (NS) is a catastrophic and enigmatic childhood epilepsy, accompanied by multiple neurological impairments and neuroinflammation. Of all the infectious, environmental and psychological factors associated with NS, the major culprit is Onchocerca Volvulus (Ov)-a parasitic worm transmitted to human by blackflies. NS seems to be an 'Autoimmune Epilepsy' in light of the recent findings of deleterious autoimmune antibodies to Glutamate receptors and to Leiomodin-I in NS patients. Moreover, we recently found immunogenetic fingerprints in HLA peptide-binding grooves associate with protection or susceptibility to NS. Macrophage migration inhibitory factor (MIF) is an immune-regulatory cytokine playing a central role in modulating innate and adaptive immunity. MIF is also involved in various pathologies: infectious, autoimmune and neurodegenerative diseases, epilepsy and others. Herein, two functional polymorphisms in the MIF gene, a -794 CATT5-8 microsatellite repeat and a -173 G/C single-nucleotide polymorphism, were assessed in 49 NS patients and 51 healthy controls from South Sudan. We also measured MIF plasma levels in established NS patients and healthy controls. We discovered that the frequency of the high-expression MIF -173C containing genotype was significantly lower in NS patients compared to healthy controls. Interestingly however, MIF plasma levels were significantly elevated in NS patients than in healthy controls. We further demonstrated that the HLA protective and susceptibility associations are dominant over the MIF association with NS. Our findings suggest that MIF might have a dual role in NS. Genetically controlled high-expression MIF genotype is associated with disease protection. However, elevated MIF in the plasma may contribute to the detrimental autoimmunity, neuroinflammation and epilepsy.

Protection or susceptibility to devastating childhood epilepsy: Nodding Syndrome associates with immunogenetic fingerprints in the HLA binding groove.

Nodding syndrome (NS) is a devastating and enigmatic childhood epilepsy. NS is accompanied by multiple neurological impairments and neuroinflammation, and associated with the parasite Onchocerca volvulus (Ov) and other environmental factors. Moreover, NS seems to be an 'Autoimmune Epilepsy' since: 1. ~50% of NS patients have neurotoxic cross-reactive Ov/Leimodin-I autoimmune antibodies. 2. Our recently published findings: Most (~86%) of NS patients have glutamate-receptor AMPA-GluR3B peptide autoimmune antibodies that bind, induce Reactive Oxygen Species, and kill both neural cells and T cells. Furthermore, NS patient's IgG induce seizures, brain multiple damage alike occurring in brains of NS patients, and elevation of T cells and activated microglia and astrocytes, in brains of normal mice. Human Leukocyte antigen (HLA) class I and II molecules are critical for initiating effective beneficial immunity against foreign microorganisms and contributing to proper brain function, but also predispose to detrimental autoimmunity against self-peptides. We analyzed seven HLA loci, either by next-generation-sequencing or Sequence-Specific-Oligonucleotide-Probe, in 48 NS patients and 51 healthy controls from South Sudan. We discovered that NS associates significantly with both protective HLA haplotype: HLA-B*42:01, C*17:01, DRB1*03:02, DQB1*04:02 and DQA1*04:01, and susceptible motif: Ala24, Glu63 and Phe67, in the HLA-B peptide-binding groove. These amino acids create a hydrophobic and sterically closed peptide-binding HLA pocket, favoring proline residue. Our findings suggest that immunogenetic fingerprints in HLA peptide-binding grooves tentatively associate with protection or susceptibility to NS. Accordingly, different HLA molecules may explain why under similar environmental factors, only some children, within the same families, tribes and districts, develop NS, while others do not.

MIF -173G/C polymorphism is associated with NMO disease severity.

Our knowledge about genetic factors that drive the worsening of neuromyelitis optica (NMO) is limited. Herein, we analyzed the macrophage migration inhibitory factor (MIF) -173G/C functional polymorphism in NMO patients and controls. Our data reveal that the frequency of the high-expression MIF genotypes (CC/GC) did not differ between the two groups. However, frequency of this genotypes was elevated in patients diagnosed with both optic neuritis and myelitis compared with patients that were diagnosed with only one symptom. Furthermore, patients carrying the CC/CG genotypes had significantly higher disability score. We conclude that MIF is associated with NMO severity rather than susceptibility.

Identification of the novel HLA-B allele, HLA-B*15:539, in a South-Sudanese individual.

HLA-B*15:539 differs from HLA-B*15:151 by two nucleotide substitutions.

Increased occurrence of anti-AQP4 seropositivity and unique HLA Class II associations with neuromyelitis optica (NMO), among Muslim Arabs in Israel.

BACKGROUND: Previous studies have revealed different human leukocyte antigen (HLA) associations in multiple sclerosis (MS) and neuromyelitis optica (NMO), further discriminating these two demyelinating pathological conditions. In worldwide analyses, NMO and opticospinal MS are represented at higher proportions among demyelinating conditions in African, East-Asian and Latin American populations. There are currently no data regarding the prevalence of NMO in Middle East Muslims. The population in Israel is diverse in many ways, and includes subpopulations, based on religion and ethnicity; some exhibit genetic homogeneity. In Israel, the incidence of MS is lower in the Muslim population than the Jewish population and Muslims carry different allele frequency distribution of HLA haplotypes. OBJECTIVE: To evaluate the occurrence of anti-AQP4 seropositivity in the Israeli Muslim population among patients with central nervous system (CNS) demyelinating conditions; and to identify the HLA DR and DQ profiles of Muslim Arab Israeli patients with NMO spectrum of diseases (NMOSD). METHODS: The prevalence of anti-AQP4 seropositivity was analyzed in 342 samples, obtained from patients with various CNS demyelinating conditions and in a validation set of 310 samples. HLA class II alleles (HLA-DRB1 and DQB1) were examined in DNA samples from 35 Israeli Muslim Arabs NMO patients and compared to available data from 74 Israeli Muslim controls. RESULTS: Our data reveal a significantly increased prevalence of anti-AQP4 seropositivity, indicative of NMOSD, in Muslim Arab Israeli patients with initial diagnosis of a CNS demyelinating syndrome. In this population, there was a positive association with the HLA-DRB1*04:04 and HLA-DRB1*10:01 alleles (p=0.03), and a strong negative association with the HLA-DRB1*07 and HLA-DQB1*02:02 alleles (p=0.003, p=0.002). CONCLUSIONS: Our findings indicate a possibly increased prevalence of NMOSD in Muslim Arabs in Israel with distinct (positive and negative) HLA associations. Further studies in patients with similar genetic backgrounds worldwide could help to confirm our findings and identify more genetic susceptibility factors for NMO, contributing to our general understanding of the pathogenesis of NMOSD.

Poly(ethylene glycol)-paclitaxel-alendronate self-assembled micelles for the targeted treatment of breast cancer bone metastases.

Paclitaxel (PTX) and alendronate (ALN) are effective drugs used for the treatment of breast cancer bone metastases. Growing evidence suggests that low-dose taxanes and bisphosphonates possess anti-angiogenic properties. However, PTX is water-insoluble and toxic, even if administered at anti-angiogenic dosing schedule. Polymer conjugation of PTX will increase water-solubility and improve its pharmacokinetic profile directing it to the tumor site. We further propose to combine it with ALN for active bone targeting. We conjugated ALN and PTX with poly(ethylene glycol) (PEG) forming self-assembled micelles where PTX molecules are located at the inner core and the water-soluble ALN molecules at the outer shell. PTX-PEG-ALN micelles exhibited similar in vitro cytotoxic and anti-angiogenic activity as the free drugs. Biodistribution analysis demonstrated preferential tumor accumulation of FITC-labeled PTX-PEG-ALN micelles. Pharmacokinetic studies revealed longer t1/2 of the conjugate than free PTX. PTX-PEG-ALN micelles achieved improved efficacy and safety profiles over free PTX in syngeneic and xenogeneic mouse models of mCherry-infected mammary adenocarcinoma in the tibia, as monitored intravitally non-invasively by a fluorescence imaging system. The described data warrants the potential use of PTX-PEG-ALN as bone-targeted anticancer and anti-angiogenic therapy for breast cancer bone metastases.

Dendritic poly(ethylene glycol) bearing paclitaxel and alendronate for targeting bone neoplasms.

Poly(ethylene glycol) (PEG) is the most popular polymer for protein conjugation, but its potential as carrier of low molecular weight drugs has been limited by the intrinsic low loading, owing to its chemical structure. In fact, only the two end chain groups of PEG can be modified and exploited for drug coupling. We have demonstrated that by synthesizing a dendrimer structure at the polymer end chains, it is possible to increase the drug payload and overcome this limitation. Furthermore, this approach can be improved by using heterobifunctional PEG. These polymers allow the precise linking of two different drugs, or a drug and a targeting agent, on the same polymeric chain. Heterobifunctional PEG-dendrimers have been obtained with defined chemical structures leading to their attractive use as drug delivery systems. In fact, they offer a double benefit; first, the possibility to choose the best drug/targeting agent ratio, and second, the separation of the two functions, activity and targeting, which are coupled at the opposite polymer end chains. In this study, we investigated the role of a PEG-dendrimer, H(2)N-PEG-dendrimer-(COOH)(4), as carrier for a combination of paclitaxel (PTX) and alendronate (ALN). PTX is a potent anticancer drug that is affected by severe side effects originating from both the drug itself and its solubilizing formulation, Cremophor EL. ALN is an aminobiphosphonate used for the treatment of osteoporosis and bone metastases as well as a bone-targeting moiety. The PTX-PEG-ALN conjugate was designed to exploit active targeting by the ALN molecule and passive targeting through the enhanced permeability and retention (EPR) effect. Our conjugate demonstrated a great binding affinity to the bone mineral hydroxyapatite in vitro and an IC(50) comparable to that of the free drugs combination in human adenocarcinoma of the prostate (PC3) cells. The PTX-PEG-ALN conjugate exhibited an improved pharmacokinetic profile compared with the free drugs owed to the marked increase in their half-life. In addition, PTX-PEG-ALN could be solubilized directly in physiological solutions without the need for Cremophor EL. The data presented in this manuscript encourage further investigations on the potential of PTX-PEG-ALN as treatment for cancer bone metastases.

Antiangiogenic antitumor activity of HPMA copolymer-paclitaxel-alendronate conjugate on breast cancer bone metastasis mouse model.

Polymer therapeutics have shown promise as tumor-targeted drug delivery systems in mice. The multivalency of polymers allows the attachment of different functional agents to a polymeric backbone, including chemotherapeutic and antiangiogenic drugs, as well as targeting moieties, such as the bone-targeting agent alendronate (ALN). We previously reported the conjugation of ALN and the chemotherapeutic drug paclitaxel (PTX) with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. The in vitro physicochemical properties, cancer cytotoxicity and antiangiogenic activity of HPMA copolymer-PTX-ALN conjugate were extensively characterized. The reported results warranted in vivo evaluations of the conjugate. In this manuscript, we evaluated the in vivo anticancer and antiangiogenic activity of HPMA copolymer-PTX-ALN conjugate. The conjugate exhibited an antiangiogenic effect by decreasing microvessel density (MVD), and inducing apoptotic circulating endothelial cells (CEC) following treatment of the mice. Using intravital imaging system and mCherry-labeled breast cancer cell lines, we were able to monitor noninvasively the progression of orthotopic metastatic tumors injected into the tibia of the mice. HPMA copolymer-PTX-ALN conjugate showed the greatest antitumor efficacy on mCherry-labeled 4T1 mammary adenocarcinoma inoculated into the tibia, as compared with PTX alone or in combination with ALN. Treatment with the bone-targeted polymeric conjugate demonstrated improved efficacy, was better tolerated, and was more easily administered intravenously than the clinically used PTX formulated in Cremophor/ethanol.

Integrin-assisted drug delivery of nano-scaled polymer therapeutics bearing paclitaxel.

Angiogenesis plays a prominent role in cancer progression. Anti-angiogenic therapy therefore, either alone or in combination with conventional cytotoxic therapy, offers a promising therapeutic approach. Paclitaxel (PTX) is a widely-used potent cytotoxic drug that also exhibits anti-angiogenic effects at low doses. However, its use, at its full potential, is limited by severe side effects. Here we designed and synthesized a targeted conjugate of PTX, a polymer and an integrin-targeted moiety resulting in a polyglutamic acid (PGA)-PTX-E-[c(RGDfK)(2)] nano-scaled conjugate. Polymer conjugation converted PTX to a macromolecule, which passively targets the tumor tissue exploiting the enhanced permeability and retention effect, while extravasating via the leaky tumor neovasculature. The cyclic RGD peptidomimetic enhanced the effects previously seen for PGA-PTX alone, utilizing the additional active targeting to the α(v)ß(3) integrin overexpressed on tumor endothelial and epithelial cells. This strategy is particularly valuable when tumors are well-vascularized, but they present poor vascular permeability. We show that PGA is enzymatically-degradable leading to PTX release under lysosomal acidic pH. PGA-PTX-E-[c(RGDfK)(2)] inhibited the growth of proliferating α(v)ß(3)-expressing endothelial cells and several cancer cells. We also showed that PGA-PTX-E-[c(RGDfK)(2)] blocked endothelial cells migration towards vascular endothelial growth factor; blocked capillary-like tube formation; and inhibited endothelial cells attachment to fibrinogen. Orthotopic studies in mice demonstrated preferential tumor accumulation of the RGD-bearing conjugate, leading to enhanced anti-tumor efficacy and a marked decrease in toxicity as compared with free PTX-treated mice.

Toward development of targeted nonsteroidal antiandrogen-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-gadolinium complex for prostate cancer diagnostics.

Androgen receptors are present in most advanced prostate cancer specimens, having a critical role in development of this type of cancer. For correct prognosis of patient conditions and treatment monitoring, noninvasive imaging techniques have great advantages over surgical procedures. We developed synthetic methodologies for preparation of novel androgen receptor-targeting agents in an attempt to build a versatile platform for prostate cancer imaging and treatment. The structure of these compounds comprises of a lanthanoid metal ion, gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Gd-DOTA)-based binding fragment and, connected to it by a flexible linker, bicalutamide-derived nonsteroidal antiandrogen moiety. A representative gadolinium complex 15 was evaluated as a magnetic resonance imaging (MRI) agent in C57/bl6 male mouse bearing orthotopic TRAMP C2 prostate tumor.

Enhanced cytotoxicity of a polymer-drug conjugate with triple payload of paclitaxel.

The development of targeting approaches to selectively release chemotherapeutic drugs into malignant tissue is a major challenge in anticancer therapy. We have synthesized an N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-drug conjugate with an AB(3) self-immolative dendritic linker. HPMA copolymers are known to accumulate selectively in tumors. The water-soluble polymer-drug conjugate was designed to release a triple payload of the hydrophobic drug paclitaxel as a result of cleavage by the endogenous enzyme cathepsin B. The polymer-drug conjugate exhibited enhanced cytotoxicity on murine prostate adenocarcinoma (TRAMP C2) cells in comparison to a classic monomeric drug-polymer conjugate.

Targeting bone metastases with a bispecific anticancer and antiangiogenic polymer-alendronate-taxane conjugate.

A polymer therapeutic designed for combination anticancer and antiangiogenic therapy inhibited the proliferation of prostate carcinoma cells and the proliferation, migration, and tube-formation of endothelial cells. The nanoconjugate was formed from an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, the bisphosphonate alendronate (for bone targeting), and the chemotherapy agent paclitaxel (PTX), which is cleaved by cathepsin B (see scheme).

E-selectin regulates gene expression in metastatic colorectal carcinoma cells and enhances HMGB1 release.

Extravasation of cancer cells is a pivotal step in the formation of hematogenous metastasis. Extravasation is initiated by the loose adhesion of cancer cells to endothelial cells via an interaction between endothelial selectins and selectin ligands expressed by the tumor cells. The present study shows that the interaction between recombinant E-selectin (rE-selectin) and colorectal cancer (CRC) cells alters the gene expression profile of the cancer cells. A DNA microarry analysis indicated that E-selectin-mediated alterations were significantly more pronounced in the metastatic CRC variants SW620 and KM12SM than in the corresponding non-metastatic local SW480 and KM12C variants. The number of genes altered by E-selectin in the metastatic variants was about 10-fold higher than the number of genes altered in the corresponding local variants. Aiming to identify genes involved in CRC metastasis, we focused, by using a DNA microarry analysis, on genes that were altered by E-selectin in a similar fashion exclusively in both metastatic variants. This analysis indicated that E-selectin down regulated (at least by 1.6-folds) the expression of 7 genes in a similar fashion, in both metastatic cells. The DNA microarry analysis was validated by real time PCR or by RT-PCR. HMGB1 was among these genes. Confocal microscopy indicated that E-selectin down regulated the cellular expression of the HMGB1 protein and enhanced the release of HMGB1 into the culture medium. The released HMGB1 in turn, activated endothelial cells to express E-selectin.