Glatiramer acetate increases T- and B -regulatory cells and decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) in an animal model of multiple sclerosis.

To identify the mechanisms relevant for the therapeutic effect of glatiramer acetate (GA), we studied T- and B- regulatory cells as well as GM-CSF expression in mice recovered from experimental autoimmune encephalomyelitis (EAE). Selective depletion of Tregs reduced but did not eliminate the ability of GA to ameliorate EAE, indicating a role for additional immune-subsets. The prevalence of Bregs in the periphery and the CNS of EAE-mice increased following GA-treatment. Furthermore, GA downregulated the pathological expression of GM-CSF, on both the protein and mRNA levels. These findings corroborate the broad immunomodulatory mechanism of action of GA in EAE/MS.

Inhibition of a pancreatic cancer model by cooperative pairs of clinically approved and experimental antibodies.

By year 2025 pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer related death. However, other than improved chemotherapy and a small molecule inhibitor of the epidermal growth factor receptor (EGFR), no targeted drugs are currently available. Repurposing of approved drugs might offer a rapid solution. We employed an animal PDAC model, expressing a mutant and a wild type form of p53 and KRAS, respectively. Cetuximab, a clinically approved anti-EGFR monoclonal antibody (mAb) weakly inhibited PDAC xenografts, similar to trastuzumab, a mAb against HER2, a co-receptor of EGFR. Because the combination of cetuximab and trastuzumab only moderately enhanced the anti-tumor effects, we combined each with a home-made mAb to the same receptor and identified two cooperative pairs. The pair of trastuzumab and a murine anti-HER2 mAb better than the anti-EGFR pair inhibited PDAC xenografts, although HER2's abundance in our model is 15-fold lower than the level of EGFR. In vitro studies attribute cooperation to forced receptor endocytosis/degradation and inhibition of both DNA synthesis and cell migration. Taken together, our results identify cooperative pairs of anti-PDAC antibodies and highlight potential mechanisms of anti-tumor effects.

Cognitive impairment in an animal model of multiple sclerosis and its amelioration by glatiramer acetate.

The severe motor impairment in the MS animal model experimental autoimmune encephalomyelitis (EAE) obstructs the assessment of cognitive functions. We developed an experimental system that evaluates memory faculties in EAE-affected mice, irrespective of their motor performance, enabling the assessment of cognitive impairments along the disease duration, the associated brain damage, and the consequences of glatiramer acetate (GA) treatment on these manifestations. The delayed-non-matching to sample (DNMS) T-maze task, testing working and long term memory was adapted and utilized. Following the appearance of clinical manifestations task performances of the EAE-untreated mice drastically declined. Cognitive impairments were associated with disease severity, as indicated by a significant correlation between the T-maze performance and the clinical symptoms in EAE-untreated mice. GA-treatment conserved cognitive functions, so that despite their exhibited mild motor impairments, the treated mice performed similarly to naïve controls. The cognitive deficit of EAE-mice coincided with inflammatory and neurodegenerative damage to the frontal cortex and the hippocampus; these damages were alleviated by GA-treatment. These combined findings indicate that in addition to motor impairment, EAE leads to substantial impairment of cognitive functions, starting at the early stages and increasing with disease aggravation. GA-treatment, conserves cognitive capacities and prevents its disease related deterioration.

Astrocyte disruption of neurovascular communication is linked to cortical damage in an animal model of multiple sclerosis.

To elucidate mechanisms contributing to cortical pathology in multiple sclerosis (MS), we investigated neurovascular aberrations, in particular the association of astrocytes with cortical neurons and blood vessels, in mice induced with experimental autoimmune encephalomyelitis (EAE). Blood-brain barrier (BBB) dysfunction was evident by leakage of the tracer sodium fluorescein, along with reduced expression of claudin-5 by endothelial cells and desmin by pericytes. Immunohistological and ultrastructural analyses revealed detachment of the astroglial cell bodies from the blood vessels and loss of their connections with both the blood vessels and the neuronal synapses. Furthermore, examination of individual astrocytic processes at cortical layer IV, where well-defined neuronal columns (barrels) are linked to functional properties, revealed loss of astrocytic confinement to the functional neuronal boundaries. Thus, in contrast to the highly modulated patches of astrocyte processes in naïve mice overlapping the barrel cores, in EAE-mice process distribution was uniform ignoring the barrel boundaries. These aberrations are attributed to the surrounding inflammation, indicated by T-cells presence in the cortex as well as in the subcortical white matter and the meninges. Immunomodulatory treatment with glatiramer acetate partially abrogated the neurovascular damage. These combined findings indicate that under inflammatory conditions, activated perivascular astrocytes fail in neuro-hemodynamic coupling, resulting in obstructed cross-talk between the blood vessels and the neurons. We propose that loss of cortical astrocytic regulation and fine-tuning between the blood supply and the neuronal needs contributes to the neurological impairment and cognitive decline occurring in EAE/MS as well as to the disease progression.

Assessing remyelination - metabolic labeling of myelin in an animal model of multiple sclerosis.

The lack of biomarkers is a major obstacle for investigating myelin repair. We used metabolic incorporation of the choline analog - propargyl-choline (P-Cho) to label and visualize newly synthesized myelin in the CNS of mice induced with experimental autoimmune encephalomyelitis (EAE). We further developed unbiased colocalization analysis to quantify P-Cho incorporation specifically into the myelin. Our findings indicate that P-Cho injection to mice recovering from EAE, either spontaneously or following glatiramer acetate treatment, results in significant elevation of its incorporation into the myelin, offering a novel strategy for assessing remyelination in animal models and the remyelination potential of candidate drugs.

Immunotherapy of cancer: from monoclonal to oligoclonal cocktails of anti-cancer antibodies: IUPHAR Review 18.

Antibody-based therapy of cancer employs monoclonal antibodies (mAbs) specific to soluble ligands, membrane antigens of T-lymphocytes or proteins located at the surface of cancer cells. The latter mAbs are often combined with cytotoxic regimens, because they block survival of residual fractions of tumours that evade therapy-induced cell death. Antibodies, along with kinase inhibitors, have become in the last decade the mainstay of oncological pharmacology. However, partial and transient responses, as well as emergence of tumour resistance, currently limit clinical application of mAbs. To overcome these hurdles, oligoclonal antibody mixtures are being tested in animal models and in clinical trials. The first homo-combination of two mAbs, each engaging a distinct site of HER2, an oncogenic receptor tyrosine kinase (RTK), has been approved for treatment of breast cancer. Likewise, a hetero-combination of antibodies to two distinct T-cell antigens, PD1 and CTLA4, has been approved for treatment of melanoma. In a similar vein, additive or synergistic anti-tumour effects observed in animal models have prompted clinical testing of hetero-combinations of antibodies simultaneously engaging distinct RTKs. We discuss the promise of antibody cocktails reminiscent of currently used mixtures of chemotherapeutics and highlight mechanisms potentially underlying their enhanced clinical efficacy.

Multimerization of ERBB2/HER2 specific aptamer leads to improved receptor binding.

Aptamers represent a promising new treatment modality for cancer. Specificity and high affinity are two parameters that characterize aptamers. In this work, we elucidated physicochemical parameters of an ERBB2/HER2 specific aptamer and determined an optimal multimerization state, leading to higher binding and improved avidity. We applied biochemical, immunochemical and biophysical methodologies to characterize binding behaviors of multimerized versions of an ERBB2/HER2 specific aptamer and demonstrate structural integrity. Finally, we show that the trimeric ERBB2/HER2 specific aptamer instigates no immunogenic response in vivo. In summary, the set of methodologies we employed establishes a way to enhance activity of a model HER2-aptamer.

Aptamer Targeting the ERBB2 Receptor Tyrosine Kinase for Applications in Tumor Therapy.

Aptamers are an emerging class of molecules in cancer therapy. They can be easily synthesized and are considered cost-effective drug candidates. In this book chapter we describe the selection and characterization of DNA aptamers specific to the human epidermal growth factor receptor 2 (ERBB2/HER2), an oncogenic tyrosine kinase. First, a DNA aptamer library is applied and ERBB2-specific aptamers are selected using SELEX. Binders are subcloned into a pGEM-T vector, sequenced, and characterized using biochemical and cell biological techniques. By multimerizing the selected ERBB2 aptamers, it might be possible to significantly increase their avidity. For example, we could show that a trimeric ERBB2-specific aptamer could efficiently internalize membranal ERBB2. Furthermore, the receptor assembled in cytoplasmic puncta and was finally degraded by the lysosome. In addition, the selected, trimeric aptamer inhibited proliferation in an XTT assay in comparison to a control sequence. Aptamers selected using the protocol we describe might exert anticancer effect. In our example of a trimeric anti-HER2 aptamer, we could report that a human gastric xenograft mouse model demonstrated pharmacological value of the selected aptamer in vivo. This chapter should enable the interested reader to replicate selection of DNA aptamers specific to oncogenic cell surface. We would like to particularly emphasize some experimental approaches which were used to further characterize selected aptamer sequences, upon SELEX selection. For instance, we included several blotting techniques, antiproliferative assays of aptamers in vitro, and describe the handling of an in vivo human xenograft mouse model.

Cancer Immunotherapy: More Is (Much) Better.

Although antibodies against EGFR and HER2 are used to treat cancer, only some patients respond and resistance often emerges. Jacobsen and colleagues present in this issue experimental evidence favoring replacement of the currently applied monoclonal antibodies with oligoclonal mixtures of six synergistic antibodies, simultaneously engaging EGFR, HER2, and also HER3.

Examination of HER3 targeting in cancer using monoclonal antibodies.

The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells' ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma-tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.

Oligodendrogenesis and myelinogenesis during postnatal development effect of glatiramer acetate.

Myelinogenesis in the mammal nervous system occurs predominantly postnatally. Glatiramer acetate (GA), a drug for the treatment for multiple sclerosis (MS), has been shown to induce immunomodulation and neuroprotection in the inflamed CNS in MS and in experimental autoimmune encephalomyelitis (EAE). Here we investigated whether GA can affect myelinogenesis and oligodendrogenesis in the developing nervous system under nonpathological conditions. Towards this end we studied myelination in mice injected daily by GA, at postnatal Days 7-21. Immunohistological and ultrastructural analyses revealed significant elevation in the number of myelinated axons as well as in the thickness of the myelin encircling them and their resulting g-ratios, in spinal cords of GA-injected mice compared with their PBS-injected littermates, at postnatal Day 14. Elevation in myelinated axons was detected also in the peripheral ventral roots of the motor nerves. GA induced also an increase in axonal diameter, implying an effect on the overall development of the nervous system. A prominent elevation in the amount of progenitor oligodendrocytes and their BrdU incorporation, as well as in mature oligodendrocytes indicated that the effect of GA is linked to increased proliferation and differentiation along the oligodendroglial maturation cascade. In addition, elevation in insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF) was found in the white matter of the GA-injected mice. Furthermore, a functional advantage in rotating rod test was exhibited by GA-injected mice over their littermates at postnatal Day 21. These cumulative findings corroborate the beneficial effect of GA on oligodendrogenesis and myelination.

Inhibition of pancreatic carcinoma by homo- and heterocombinations of antibodies against EGF-receptor and its kin HER2/ErbB-2.

Due to intrinsic aggressiveness and lack of effective therapies, prognosis of pancreatic cancer remains dismal. Because the only molecular targeted drug approved for pancreatic ductal adenocarcinoma is a kinase inhibitor specific to the epidermal growth factor receptor (EGFR), and this receptor collaborates with another kinase, called HER2 (human EGF-receptor 2), we assumed that agents targeting EGFR and/or HER2 would effectively retard pancreatic ductal adenocarcinoma. Accordingly, two immunological strategies were tested in animal models: (i) two antibodies able to engage distinct epitopes of either EGFR or HER2 were separately combined, and (ii) pairs of one antibody to EGFR and another to HER2. Unlike the respective single monoclonal antibodies, which induced weak effects, both types of antibody combinations synergized in animals in terms of tumor inhibition. Immunological cooperation may not depend on receptor density, antigenic sites, or the presence of a mutant RAS protein. Nevertheless, both types of antibody combinations enhanced receptor degradation. Future efforts will examine the feasibility of each strategy and the potential of combining them to achieve sustained tumor inhibition.

Antibiotic susceptibility of Aggregatibacter actinomycetemcomitans JP2 in a biofilm.

BACKGROUND: Localized aggressive periodontitis (LAgP) is an inflammatory disease associated with specific bacteria, particularly Aggregatibacter actinomycetemcomitans, which can result in early tooth loss. The bacteria grow as a biofilm known as subgingival plaque. Treatment includes mechanical debridement of the biofilm, often associated with empirical antibiotic treatment. OBJECTIVE: The aims of this study were to test in vitro the sensitivity of A. actinomycetemcomitans JP2 during planktonic and biofilm growth to doxycycline and to the combination of metronidazole and amoxicillin, which are two antibiotic protocols commonly used in clinical practice. DESIGN: Two in vitro biofilm models were used to test the effects of the antibiotics: a static 96-well plate assay was used to investigate the effect of these antibiotics on biofilm formation whilst a flow chamber model was used to examine the effect on established biofilms. RESULTS: Of the antibiotics tested in this model system, doxycycline was most efficacious with a minimal inhibitory concentration (MIC) against planktonic cells of 0.21 mg/L and minimal biofilm inhibitory concentration (MBIC) of 2.10 mg/L. The most commonly prescribed antibiotic regimen, amoxicillin + metronidazole, was much less effective against both planktonic and biofilm cells with an MIC and MBIC of 12.0 mg/L and 20.2 mg/L, respectively. A single treatment of the clinically achievable concentration of 10 mg/L doxycycline to sparse A. actinomycetemcomitans biofilms in the flow chamber model resulted in significant decreases in biofilm thickness, biovolume, and cell viability. Dense A. actinomycetemcomitans biofilms were significantly more resistant to doxycycline treatment. Low concentrations of antibiotics enhanced biofilm formation. CONCLUSION: A. actinomycetemcomitans JP2 homotypic biofilms were more susceptible in vitro to doxycycline than amoxicillin + metronidazole.

Aptamer to ErbB-2/HER2 enhances degradation of the target and inhibits tumorigenic growth.

Aptamers, oligonucleotides able to avidly bind cellular targets, are emerging as promising therapeutic agents, analogous to monoclonal antibodies. We selected from a DNA library an aptamer specifically recognizing human epidermal growth factor receptor 2 (ErbB-2/HER2), a receptor tyrosine kinase, which is overexpressed in a variety of human cancers, including breast and gastric tumors. Treatment of human gastric cancer cells with a trimeric version (42 nucleotides) of the selected aptamer (14 nucleotides) resulted in reduced cell growth in vitro, but a monomeric version was ineffective. Likewise, when treated with the trimeric aptamer, animals bearing tumor xenografts of human gastric origin reflected reduced rates of tumor growth. The antitumor effect of the aptamer was nearly twofold stronger than that of a monoclonal anti-ErbB-2/HER2 antibody. Consistent with aptamer-induced intracellular degradation of ErbB-2/HER2, incubation of gastric cancer cells with the trimeric aptamer promoted translocation of ErbB-2/HER2 from the cell surface to cytoplasmic puncta. This translocation was associated with a lysosomal hydrolase-dependent clearance of the ErbB-2/HER2 protein from cell extracts. We conclude that targeting ErbB-2/HER2 with DNA aptamers might retard the tumorigenic growth of gastric cancer by means of accelerating lysosomal degradation of the oncoprotein. This work exemplifies the potential pharmacological utility of aptamers directed at cell surface proteins, and it highlights an endocytosis-mediated mechanism of tumor inhibition.

Inhibition of triple-negative breast cancer models by combinations of antibodies to EGFR.

Breast tumors lacking expression of human epidermal growth factor receptor 2 (HER2) and the estrogen and the progesterone receptors (triple negative; TNBC) are more aggressive than other disease subtypes, and no molecular targeted agents are currently available for their treatment. Because TNBC commonly displays EGF receptor (EGFR) expression, and combinations of monoclonal antibodies to EGFR effectively inhibit other tumor models, we addressed the relevance of this strategy to treatment of TNBC. Unlike a combination of the clinically approved monoclonal antibodies, cetuximab and panitumumab, which displaced each other and displayed no cooperative effects, several other combinations resulted in enhanced inhibition of TNBC's cell growth both in vitro and in animals. The ability of certain antibody mixtures to remove EGFR from the cell surface and to promote its intracellular degradation correlated with the inhibitory potential. However, unlike EGF-induced sorting of EGFR to lysosomal degradation, the antibody-induced pathway displayed independence from the intrinsic kinase activity and dimer formation ability of EGFR, and it largely avoided the recycling route. In conclusion, although TNBC clinical trials testing EGFR inhibitors reported lack of benefit, our results offer an alternative strategy that combines noncompetitive antibodies to achieve robust degradation of EGFR and tumor inhibition.

The adhesion of oral bacteria to modified titanium surfaces: role of plasma proteins and electrostatic forces.

OBJECTIVES: Modifications of titanium (Ti) implant surfaces have a significant effect on early biofilm formation and the outcome of implant procedures. The aim of this study was to examine the role of plasma proteins and electrostatic forces in the adhesion mechanism of oral bacteria to modified Ti surfaces. MATERIALS AND METHODS: Ti discs with three different types of surface modifications, machined, acid-etched, and acid-etched and blasted, were examined for adhesion of oral bacteria: Streptococcus mutans, Porphyromonas gingivalis, and Fusobacterium nucleatum. Following pretreatment of the Ti with ion rich solutions or coating by human serum albumin or fibronectin, bacterial adhesion was examined by scanning electron microscopy and assessed quantitatively by DNA analysis. Ti coating by proteins as well as bacterial adhesion and their interrelationships were further investigated through confocal scanning laser microscopy. RESULTS: Acid-etched and blasted Ti surfaces exhibited significantly higher amounts of bacteria adhesion than the other two surfaces. Calcium was found to serve as a bridging agent in the adhesion process of S. mutans and F. nucleatum to Ti surfaces. Although albumin coating of the Ti reduced the adhesion of S. mutans to all surfaces, it had no influence on the adhesion of P. gingivalis or F. nucleatum. Coating the Ti with fibronectin enhanced P. gingivalis and F. nucleatum adhesion. CONCLUSIONS: Bacterial adhesion to Ti surfaces is roughness-dependent, and the adhesion mechanism is influenced by ions and proteins of the initial coating derived from the blood.

Wettability versus electrostatic forces in fibronectin and albumin adsorption to titanium surfaces.

OBJECTIVES: Although the enhancement of plasma protein adsorption to titanium ( Ti ) following wetting has been recognized, the relationship between wettability and electrostatic forces has remained unclear. Thus, we have carried out a series of studies to determine the role of wettability and electrostatic forces on protein adsorption. METHODS: Titanium disks with different surfaces were wetted with a range of solutions, two of which contained divalent positive ions ( Ca and Mg ). Unwetted disks served as a control. Subsequently, the wetted disks were subjected to three treatment regimes: (1) incubation in human serum albumin (HSA) or human serum fibronectin (HSF); (2) drying the wetted disks, followed by incubation in HSA or HSF; and (3) following protein adsorption, the Ca originating in the wetting solutions was removed by divalent positive ions chelator treatment (EGTA), and the remaining quantities were assessed. The quantity of the adsorbed proteins was determined by ELISA. RESULTS: It was found that in the case of HSA, adsorption was enhanced by the wettability, the presence of Ca and Mg in the wetting solution, and the existence of rough surfaces. For HSF, the wettability and rough surfaces enhanced adsorption. CONCLUSION: The results demonstrate that in addition to wettability, the composition of the wetting solution affects the protein adsorption. While wetting reduces the time for the HSA and HSF adsorption to reach saturation, the electrostatic forces enhance the amount of HSA adsorption. Thus, the protein adsorption capacity of titanium rough surfaces can be selectively manipulated by changing of the wetting solution.