Pyridoxine or pyridoxal-5-phosphate treatment for seizures in glycosylphosphatidylinositol deficiency: A cohort study.

AIM: To investigate the short-term efficacy and safety of high-dose pyridoxine and pyridoxal 5-phosphate (P5P) in the treatment of inherited glycosylphosphatidylinositol (GPI) deficiency-associated epilepsy. METHOD: Participants with genetically confirmed GPI deficiency were treated with oral pyridoxine or P5P as compassionate use in an agreed-upon clinical regimen. Pyridoxine (20-30 mg/kg/day) was used for 3 months. Baseline evaluation included 4 weeks of prospective seizure data and one video electroencephalogram (EEG). Seizure frequency was captured daily. The EEG was repeated after reaching maximum dosage of pyridoxine. Pyridoxine was switched to P5P (20-30 mg/kg/day) if seizure burden was unchanged after 3 months' treatment. Another EEG was done after 3 months of P5P treatment. Primary outcome measures were reduction of seizure frequency and EEG improvements. RESULTS: Seven participants (one female, six males; age range 5-23 year; mean age 11 years 10 months, SD 5 year 2 months) were included. The genetic causes of inherited GPI deficiency were phosphatidylinositol N-acetylglucosaminyltransferase subunit A/T/V deficiency. All had drug-resistant epilepsy and neurodevelopmental impairment. We observed more than 50% seizure frequency reduction in 2 out of 7 and less than 50% reduction in another 3 out of 7 participants. No participants reached seizure freedom. No remarkable changes in electrophysiological findings were observed in 6 out of 7 participants treated with pyridoxine or P5P when comparing the baseline and follow-up EEGs. INTERPRETATION: We observed no long-lasting electrophysiological improvements during treatment but pyridoxine may reduce seizure frequency or burden in inherited GPI deficiency. WHAT THIS PAPER ADDS: Inherited glycosylphosphatidylinositol (GPI) deficiency often causes early-onset and drug-resistant epilepsy. Vitamin B6 is a potential disease-specific treatment; however, efficacy and safety are ill-defined. Pyridoxine may reduce seizure frequency or burden in inherited GPI deficiency. Pyridoxine and P5P could prove to be a useful treatment in some individuals with inherited GPI deficiency and epilepsy.

Emerging roles for the GPI-anchored tumor suppressor OPCML in cancers.

OPCML is a highly conserved glycosyl phosphatidylinositol (GPI)-anchored protein belonging to the IgLON family of cell adhesion molecules. OPCML functions as a tumor suppressor and is silenced in over 80% of ovarian cancers by loss of heterozygosity and by epigenetic mechanisms. OPCML inactivation is also observed in many other cancers suggesting a conservation of tumor suppressor function. Although epigenetic silencing and subsequent loss of OPCML expression correlate with poor progression-free and overall patient survival, its mechanism of action is only starting to be fully elucidated. Recent discoveries have demonstrated that OPCML exerts its tumor suppressor effect by inhibiting several cancer hallmark phenotypes in vitro and abrogating tumorigenesis in vivo, by downregulating/inactivating a specific spectrum of Receptor Tyrosine Kinases (RTKs), including EphA2, FGFR1, FGFR3, HER2, HER4, and AXL. This modulation of RTKs can also sensitize ovarian and breast cancers to lapatinib, erlotinib, and anti-AXL therapies. Furthermore, OPCML has also been shown to function in synergy with the tumor suppressor phosphatase PTPRG to inactivate pro-metastatic RTKs such as AXL. Recently, the identification of inactivating point mutations and the elucidation of the crystal structure of OPCML have provided valuable insights into its structure-function relationships, giving rise to its potential as an anti-cancer therapeutic.

Malaria Derived Glycosylphosphatidylinositol Anchor Enhances Anti-Pfs25 Functional Antibodies That Block Malaria Transmission.

Malaria, one of the most common vector borne human diseases, is a major world health issue. In 2015 alone, more than 200 million people were infected with malaria, out of which, 429 000 died. Even though artemisinin-based combination therapies (ACT) are highly effective at treating malaria infections, novel efforts toward development of vaccines to prevent transmission are still needed. Pfs25, a postfertilization stage parasite surface antigen, is a leading transmission-blocking vaccine (TBV) candidate. It is postulated that Pfs25 anchors to the cell membrane using a glycosylphosphatidylinositol (GPI) linker, which itself possesses pro-inflammatory properties. In this study, Escherichia coli derived extract (XtractCF+TM) was used in cell free protein synthesis [CFPS] to successfully express >200 mg/L of recombinant Pfs25 with a C-terminal non-natural amino acid (nnAA), namely, p-azidomethyl phenylalanine (pAMF), which possesses a reactive azide group. Thereafter, a unique conjugate vaccine (CV), namely, Pfs25-GPI was generated with dibenzocyclooctyne (DBCO) derivatized glycan core of malaria GPI using a simple but highly efficient copper free click chemistry reaction. In mice immunized with Pfs25 or Pfs25-GPI, the Pfs25-GPI group showed significantly higher titers compared to the Pfs25 group. Moreover, only purified IgGs from Pfs25-GPI group were able to significantly block transmission of parasites to mosquitoes, as judged by a standard membrane feeding assay [SMFA]. To our knowledge, this is the first report of the generation of a CV using Pfs25 and malaria specific GPI where the GPI is shown to enhance the ability of Pfs25 to elicit transmission blocking antibodies.

A novel CXCL10-based GPI-anchored fusion protein as adjuvant in NK-based tumor therapy.

BACKGROUND: Cellular therapy is a promising therapeutic strategy for malignant diseases. The efficacy of this therapy can be limited by poor infiltration of the tumor by immune effector cells. In particular, NK cell infiltration is often reduced relative to T cells. A novel class of fusion proteins was designed to enhance the recruitment of specific leukocyte subsets based on their expression of a given chemokine receptor. The proteins are composed of an N-terminal chemokine head, the mucin domain taken from the membrane-anchored chemokine CX3CL1, and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor replacing the normal transmembrane domain allowing integration of the proteins into cell membranes when injected into a solid tumor. The mucin domain in conjunction with the chemokine head acts to specifically recruit leukocytes expressing the corresponding chemokine receptor. METHODOLOGY/PRINCIPAL FINDINGS: A fusion protein comprising a CXCL10 chemokine head (CXCL10-mucin-GPI) was used for proof of concept for this approach and expressed constitutively in Chinese Hamster Ovary cells. FPLC was used to purify proteins. The recombinant proteins efficiently integrated into cell membranes in a process dependent upon the GPI anchor and were able to activate the CXCR3 receptor on lymphocytes. Endothelial cells incubated with CXCL10-mucin-GPI efficiently recruited NK cells in vitro under conditions of physiologic flow, which was shown to be dependent on the presence of the mucin domain. Experiments conducted in vivo using established tumors in mice suggested a positive effect of CXCL10-mucin-GPI on the recruitment of NK cells. CONCLUSIONS: The results suggest enhanced recruitment of NK cells by CXCL10-mucin-GPI. This class of fusion proteins represents a novel adjuvant in cellular immunotherapy. The underlying concept of a chemokine head fused to the mucin domain and a GPI anchor signal sequence may be expanded into a broader family of reagents that will allow targeted recruitment of cells in various settings.

Chemical biology of glycosylphosphatidylinositol anchors.

Glycosylphosphatidylinositols (GPIs) are complex glycolipids that are covalently linked to the C-terminus of proteins as a posttranslational modification. They anchor the attached protein to the cell membrane and are essential for normal functioning of eukaryotic cells. GPI-anchored proteins are structurally and functionally diverse. Many GPIs have been structurally characterized but comprehension of their biological functions, beyond the simple physical anchoring, remains largely speculative. Work on functional elucidation at a molecular level is still limited. This Review focuses on the roles of GPI unraveled by using synthetic molecules and summarizes the structural diversity of GPIs, as well as their biological and chemical syntheses.

Cell surface engineering of renal cell carcinoma with glycosylphosphatidylinositol-anchored TIMP-1 blocks TGF- ß 1 activation and reduces regulatory ID gene expression.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) controls matrix metalloproteinase activity through 1:1stoichiometric binding. Human TIMP-1 fused to a glycosylphosphatidylinositol(GPI) anchor (TIMP-1 - GPI) shifts the activity of TIMP-1 from the extracellular matrix to the cell surface. TIMP-1 - GPI treated renal cell carcinoma cells show increased apoptosis and reduced proliferation.Transcriptomic profiling and regulatory pathway mapping were used to identify the potential mechanisms driving these effects. Significant changes in the DNA binding inhibitors, TGF- ß 1/SMAD and BMP pathways resulted from TIMP-1 - GPI treatment. These events were linked to reduced TGF- ß 1 signaling mediated by inhibition of proteolytic processing of latent TGF- ß 1 by TIMP-1 - GPI.

The central role of macrophages in trypanosomiasis-associated anemia: rationale for therapeutical approaches.

Bovine African trypanosomiasis causes severe economical problems on the African continent and one of the most prominent immunopathological parameters associated with this parasitic infection is anemia. In this report we review the current knowledge of the mechanisms underlying trypanosomiasis-associated anemia. In first instance, the central role of macrophages and particularly their activation state in determining the outcome of the disease (i.e. trypanosusceptibility versus trypanotolerance) will be discussed. In essence, while persistence of classically activated macrophages (M1) contributes to anemia development, switching towards alternatively activated macrophages (M2) alleviates pathology including anemia. Secondly, while parasite-derived glycolipids such as the glycosylphosphatidylinositol (GPI) induce M1, host-derived IL-10 blocks M1-mediated inflammation, promotes M2 development and prevents anemia development. In this context, strategies aimed at inducing the M1 to M2 switch, such as GPI-based treatment, adenoviral delivery of IL-10 and induction of IL-10 producing regulatory T cells will be discussed. Finally, the crucial role of iron-homeostasis in trypanosomiasis-associated anemia development will be documented to stress the analogy with anemia of chronic disease (ACD), hereby providing new insight that might contribute to the treatment of ACD.

Peritumoral administration of GPI-anchored TIMP-1 inhibits colon carcinoma growth in Rag-2 gamma chain-deficient mice.

Exogenous application of recombinant TIMP-1 protein modified by addition of a glycosylphosphatidylinositol (GPI) anchor allows efficient insertion of the fusion protein into cell membranes. This 'cell surface engineering' leads to changes in the proteolytic environment. TIMP-1-GPI shows enhanced as well as novel in vitro biological activities including suppression of proliferation, reduced migration, and inhibition of invasion of the colon carcinoma cell line SW480. Treatment of SW480 tumors implanted in Rag (-/-) common gamma chain (-/-) C57BL/6 mice with peritumorally applied TIMP-1-GPI, control rhTIMP-1 protein, or vehicle shows that TIMP-1-GPI leads to a significant reduction in tumor growth.

A glycosylphosphatidylinositol-based treatment alleviates trypanosomiasis-associated immunopathology.

The GPI-anchored trypanosome variant surface glycoprotein (VSG) triggers macrophages to produce TNF, involved in trypanosomiasis-associated inflammation and the clinical manifestation of sleeping sickness. Aiming at inhibiting immunopathology during experimental Trypanosoma brucei infections, a VSG-derived GPI-based treatment approach was developed. To achieve this, mice were exposed to the GPI before an infectious trypanosome challenge. This GPI-based strategy resulted in a significant prolonged survival and a substantial protection against infection-associated weight loss, liver damage, acidosis, and anemia; the latter was shown to be Ab-independent and correlated with reduced macrophage-mediated RBC clearance. In addition, GPI-based treatment resulted in reduced circulating serum levels of the inflammatory cytokines TNF and IL-6, abrogation of infection-induced LPS hypersensitivity, and an increase in circulating IL-10. At the level of trypanosomiasis-associated macrophage activation, the GPI-based treatment resulted in an impaired secretion of TNF by VSG and LPS pulsed macrophages, a reduced expression of the inflammatory cytokine genes TNF, IL-6, and IL-12, and an increased expression of the anti-inflammatory cytokine gene IL-10. In addition, this change in cytokine pattern upon GPI-based treatment was associated with the expression of alternatively activated macrophage markers. Finally, the GPI-based treatment also reduced the infection-associated pathology in Trypanosoma congolense and Trypanosoma evansi model systems as well as in tsetse fly challenge experiments, indicating potential field applicability for this intervention strategy.

Synthetic glycovaccine protects against the bite of leishmania-infected sand flies.

Leishmaniasis is a vectorborne disease transmitted to human and other mammalian hosts by sand fly bite. In the present study, we show that immunization with Leishmania mexicana promastigote secretory gel (PSG) or with a chemically defined synthetic glycovaccine containing the glycans found in L. mexicana PSG can provide significant protection against challenge by the bite of infected sand flies. Only the glycan from L. mexicana was protective; those from other species did not protect against L. mexicana infection. Furthermore, neither PSG nor the glycovaccine protected against artificial needle challenge, which is traditionally used in antileishmanial vaccine development. Conversely, an antigen preparation that was effective against needle challenge offered no protection against sand fly bite. These findings provide a new target for Leishmania vaccine development and demonstrate the critical role that the vector plays in the evaluation of candidate vaccines for leishmaniasis and other vectorborne diseases.

Signalling via caveolin: involvement in the cross-talk between phosphoinositolglycans and insulin.

In recent years, a number of cross-talk systems have been identified which feed into the insulin signalling cascade at the level of insulin receptor substrate (IRS) tyrosine phosphorylation, e.g., receptor and non-receptor tyrosine kinases and G-protein-coupled receptors. At the molecular level, a number of negative modulator and feedback systems somehow interacting with the beta-subunit (catecholamine-, phorbolester-, or tumor necrosis factor-alpha-induced serine/threonine phosphorylation, carboxy-terminal trimming by a thiol-dependent protease, association of inhibitory/regulatory proteins such as RAD, PC1, PED, alpha2-HS-glycoprotein) have been identified as candidate mechanisms for the impairment of insulin receptor function by elevations in the activity and/or amount of the corresponding modification enzymes/inhibitors. Both decreased responsiveness and sensitivity of the insulin receptor beta-subunit for insulin-induced tyrosine autophosphorylation have been demonstrated in several cellular and animal models of metabolic insulin resistance as well as in the adipose tissue and skeletal muscle of diabetic patients and obese Pima Indians compared to non-obese subjects. Therefore, induction of the insulin signalling cascade by bypassing the defective insulin receptor kinase may be useful for the therapy of non-insulin dependent diabetes mellitus. During the past two decades, phosphoinositolglycans (PIGs) of various origin have been demonstrated to exert potent insulin-mimetic metabolic effects upon incubation with cultured or isolated muscle and adipose cells. However, it remained to be elucidated whether these compounds actually manage to trigger insulin signalling and if so at which level of hierarchy within the signalling cascade the site of interference is located. Recent studies using isolated rat adipocytes and chemically synthesized PIG compounds point to IRS1/3 tyrosine phosphorylation by p59Lyn kinase as the site of cross-talk, the negative regulation of which by interaction with caveolin is apparently abrogated by PIG. This putative mechanism is thus compatible with the recently formulated caveolin signalling hypothesis, the supporting data for which are reviewed here. Though we have not obtained experimental evidence for the involvement of PIG in physiological insulin action, the potential cross-talk between insulin and PIG signalling, including the caveolae/detergent-insoluble glycolipid-enriched rafts as the compartments where the corresponding signalling components are concentrated, thus represent novel targets for signal transduction therapy.