Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

Optimization of capsid-incorporated antigens for a novel adenovirus vaccine approach

Background

Human rhinoviruses (HRVs) are the predominant cause of common cold. In addition, HRVs are implicated in the worsening of COPD and asthma, as well as the loss of lung transplants. Despite significant efforts, no anti-viral agent is approved for the prevention or treatment of HRV-infection.

Results

In this study we demonstrate that Iota-Carrageenan, a sulphated polysaccharide derived from red seaweed, is a potent anti-rhinoviral substance in-vitro. Iota-Carrageenan reduces HRV growth and inhibits the virus induced cythopathic effect of infected HeLa cells. In addition, Iota-Carrageenan effectively prevents the replication of HRV1A, HRV2, HRV8, HRV14, HRV16, HRV83 and HRV84 in primary human nasal epithelial cells in culture. The data suggest that Iota-Carrageenan acts primarily by preventing the binding or the entry of virions into the cells.

Conclusion

Since HRV infections predominately occur in the nasal cavity and the upper respiratory tract, a targeted treatment with a product containing Iota-Carrageenan is conceivable. Clinical trials are needed to determine whether Iota-Carrageenan-based products are effective in the treatment or prophylaxis of HRV infections.     

The convergent properties of the neocortical vestibular area in the cat]

In acute experiments on cats under nembutal-chloralose anaesthesia the evoked potentials and cellular reactions were studied of suprasylvian vestibular and auditory projection zones to stimulation of vestibular, acoustic and visual nerves and frontal paw. It has been shown that the suprasylvian vestibular zone represents the region of convergence of vestibular, auditory, somatic and visual afferentation. Properties of summary and cellular reactions of the vestibular zone and also the character of interaction of the evoked potentials of homo- and heteromodal origin testify to the absence of significant dominance of vestibular input to this area of the cerebral cortex in cats. Limitation of spreading of labyrinth activity in the cerebral cortex and the absence of dominance of homomodal input in the projection zone should, probably, be considered as typical property of the vestibular system presentation in the cortex, determining the disability of monomodal specific reaction in the sensory-perceptive sphere.     

Some features of cyclic adenosine monophosphate metabolism in mouse liver and hepatoma 22]

The levels of cyclic adenosine monophosphate (cAMP) and two forms of cAMP phosphodiesterase with low (PDE1) and high (PDE2) affinity for the substrate were determined in homogenates from mouse liver and transplanted hepatoma 22. The level of cAMP in the tumour is 3 times lower than that in liver. By te kinetic parameters (Vmax, Km, pH optimum) adenylate cyclase from tumour does not show any significant differences as compared to the liver enzyme; the enzyme from hepatoma is, however, more sensitive to activation by F- ions. The activities of adenylate cyclase in liver and tumour cells are the same. Phosphodiesterases of cAMP from tumour and liver cells are similar in their Km values (3,3-10(-4) M for PDE1 and 2-10(-6) M for PDE2); however, the maximal and real rates of cAMP hydrolysis in hepatoma are much higher than in liver. The fact that both cAMP phosphodiesterase activities have similar dependence on Mg2+ and Ca2+ concentrations, suggests that PDE1 is a latent form of PDE2. In tumour cells the equilibrium between these two forms is probably shifted towards the enzyme with high affinity for the substrate. The results suggest that a decreased cAMP level in hepatoma cells (as compared to the liver) is due to the activation of PDE2.     

Heparin-Binding Proteins in the Rat Brain

Using a histochemical technique, we found that in rat embryos heparin-binding sites are localized within ventricular regions of the neural tube. The highest intensity of the heparin-binding activity was observed in the membranes of migrating nerve cells. Heparin-binding membrane-associated proteins were isolated and purified from the brains of newborn rats; molecular masses of two such proteins were measured (19 and 28 kdalton). The level of affinity for binding of heparan sulfate to the purified proteins was characterized by equilibrium constants of 1.7 · 10^-3 and 6.7 · 10^-3. Binding of heparan sulfate to the above proteins was more intensive at low ion force and pH values within the 3.0 to 4.0 range and about 6.0.     

Characterization of an Adenovirus Vector Containing a Heterologous Peptide Epitope in the HI Loop of the Fiber Knob

The utility of the present generation of recombinant adenovirus vectors for gene therapy applications could potentially be improved by designing targeted vectors capable of gene delivery to selected cell types in vivo. In order to achieve such targeting, we are investigating the possibilities of incorporation of ligands in the adenovirus fiber protein, which mediates primary binding of adenovirus to its cell surface receptor. Based on the proposed structure of the cell-binding domain of the fiber, we hypothesized that the HI loop of the fiber knob can be utilized as a convenient locale for incorporation of heterologous ligands. In this study, we utilized recombinant fiber proteins expressed in baculovirus-infected insect cells to demonstrate that the incorporation of the FLAG octapeptide into the HI loop does not ablate fiber trimerization and does not disturb formation of the cell-binding site localized in the knob. We then generated a recombinant adenovirus containing this modified fiber and showed that the short peptide sequence engineered in the knob is compatible with the biological functions of the fiber. In addition, by using a ligand-specific antibody, we have shown that the peptide incorporated into the knob remains available for binding in the context of mature virions containing modified fibers. These findings suggest that heterologous ligands can be incorporated into the HI loop of the fiber knob and that this locale possesses properties consistent with its employment in adenovirus retargeting strategies.Recombinant adenovirus vectors have found wide employment for a number of gene therapy applications (22, 36, 40). This fact has derived principally from the high levels of gene transfer achievable with this vector approach both in vitro and in vivo. Indeed, recombinant adenovirus vectors are distinguished from other available systems by their unique ability to accomplish in situ gene delivery to differentiated target cells in a variety of organ contexts (5, 6, 9, 10, 12, 21, 26, 28, 30, 32). Despite this property, specific aspects of the adenovirus biology have prevented the full realization of the potential of such vectors. In this regard, the broad tropism profile of the parent virus for cells of diverse tissues potentially allows unrestricted gene delivery. Thus, for the many gene therapy applications requiring targeted, cell-specific gene delivery, the promiscuous tropism of the adenovirus vector represents a confounding factor. Based on this concept, strategies to modify the native tropism of adenovirus have been developed to allow the derivation of vectors capable of targeted gene delivery.Strategies to achieve this end are directed at modifying specific steps in the adenovirus infection pathway. Adenoviruses of serotypes 2 and 5 normally achieve initial recognition and binding to target cells by means of interactions between the carboxy-terminal knob domain of the fiber protein and the primary receptor (4, 19, 39). After binding, RGD motifs in the penton base interact with cellular integrins of the α_Vβ₃ and α_Vβ₅ types (1–3, 43, 44). This interaction triggers cellular internalization whereby the virions achieve localization within the endosome. Acidification of the endosome elicits conformational changes in capsid proteins, allowing their interaction with the endosome membrane in a manner that achieves vesicle disruption and particle escape (41). Following endosomolysis, the virion translocates to the nucleus, where the subsequent steps of the viral life cycle occur. This understanding of the key role played by capsid proteins in the viral infectious pathway has suggested strategies to alter this process via modifications of these proteins.In this regard, genetic retargeting of adenovirus vectors via modification of viral genes encoding coat proteins, if successful, offers a simple way to achieve a significant improvement in the present generation of these gene-delivery vehicles. To this end, several groups have reported genetic modifications to the knob domain of adenovirus fiber protein and incorporation of such chimeric fibers into virions. For instance, Stevenson et al. (37) and Krasnykh et al. (25) reported successful generation of adenovirus type 5 (Ad5) virions containing fibers consisting of the tail and shaft domains of Ad5 fiber and the knob domain of Ad3, respectively. In addition, Michael et al. (31) demonstrated the incorporation of the gastrin-releasing peptide into the carboxy terminus of recombinant Ad5 fiber. This finding was extended by Legrand et al. (30a), who achieved rescue of recombinant adenovirus vectors containing such fibers. Another report published by Wickham et al. (45) described the generation of recombinant virus containing fibers with carboxy-terminal polylysine sequences. These studies have established key feasibility issues with respect to this genetic approach but have also demonstrated a number of potentially limiting factors.Of note, all the modifications of adenovirus fiber reported so far were directed towards the carboxy terminus of the protein. In addition, these efforts were initiated without prior knowledge of the three-dimensional (3D) structure of the fiber knob. Thus, the employment of the carboxy terminus of the fiber represented a choice of convenience without consideration of the knob tertiary structure. Clearly, 3D structural information has important bearing upon the placement of heterologous protein sequences within the knob for targeting purposes. Such localization of targeting ligands would ideally be achieved in such a manner as to allow their surface presentation and to minimally perturb the fiber quaternary structure. Thus, the recent crystallization of the fiber knob by Xia et al. (47, 48) has provided a level of structural resolution potentially allowing such a rational modification of the fiber protein. According to the proposed 3D model of the knob (Fig. ​(Fig.1),1), the HI loop possesses a number of features which predict its utility as an alternative site for ligand incorporation. Specifically, the HI loop does not contribute to intramolecular interactions in the knob. Therefore, incorporation of additional protein sequence should not affect the trimerization of the fiber. In addition, the loop consists mostly of hydrophilic amino acid residues and is exposed outside the knob. It thus potentially demonstrates a high degree of flexibility, creating an optimal environment for ligand incorporation. Furthermore, the lengths of HI loops vary significantly in knobs of different adenovirus serotypes. This fact suggests that alterations of the original structure of the loop, such as insertions and deletions, should be compatible with the correct folding of the entire knob domain. Finally, the HI loop is not involved in the formation of the putative cell-binding site localized in the knob. Open in a separate windowFIG. 13D model of the Ad5 fiber knob. The trimer forms a propeller-like structure when it is viewed along the threefold-symmetry axis from above. The HI loop, exposed outside the knob, connects the β-strands H and I, which are involved in the formation of the cell-binding site. (Reproduced from reference 47 by permission.)Based on these considerations, we endeavored to develop a novel approach to modify the adenovirus fiber protein by employing the HI loop of the knob for this purpose. We show in this report that it is possible to incorporate heterologous amino acid sequences into the HI loop without affecting the correct folding of the fiber polypeptide and its biological functions. Further, our results suggest that this locale may offer advantages for strategies designed to achieve tropism modification based on genetic alteration of capsid proteins.