Structure-guided engineering of tick evasins for targeting chemokines in inflammatory diseases

As natural chemokine inhibitors, evasin proteins produced in tick saliva are potential therapeutic agents for numerous inflammatory diseases. Engineering evasins to block the desired chemokines and avoid off-target side effects requires structural understanding of their target selectivity. Structures of the class A evasin EVA-P974 bound to human CC chemokine ligands 7 and 17 (CCL7 and CCL17) and to a CCL8-CCL7 chimera reveal that the specificity of class A evasins for chemokines of the CC subfamily is defined by conserved, rigid backbone–backbone interactions, whereas the preference for a subset of CC chemokines is controlled by side-chain interactions at four hotspots in flexible structural elements. Hotspot mutations alter target preference, enabling inhibition of selected chemokines. The structure of an engineered EVA-P974 bound to CCL2 reveals an underlying molecular mechanism of EVA-P974 target preference. These results provide a structure-based framework for engineering evasins as targeted antiinflammatory therapeutics.

While some proteins exhibit absolute specificity for a unique binding partner, many others display “multispecificity,” whereby they interact with several, but not all, members of a partner protein family (1, 2). Understanding how proteins achieve such selectivity provides a basis for rational engineering to regulate alternative targets. In this study, we investigated the structural basis for multispecific recognition of human proinflammatory chemokines by tick evasin proteins.Chemokines are the master regulators of leukocyte-trafficking, the unifying feature of immune homeostasis and all inflammatory diseases (3). Chemokines stimulate leukocyte migration via activation of chemokine receptors, G protein–coupled receptors expressed on the surfaces of leukocytes. Chemokines are classified into two major families (CCL and CXCL) and two minor families (XCL and CX₃CL) based on the arrangement of conserved cysteine residues near the N termini of their amino acid sequences. Chemokine receptors are classified (CCR, CXCR, XCR, and CX₃CR) based on their chemokine selectivity. The types of leukocytes recruited to specific tissues depend on the array of chemokines expressed in those tissues and the selectivity of those chemokines for the receptors expressed on different leukocyte subsets. For example, in vascular inflammation associated with hypertension, elevated levels of the chemokines CCL2, CCL7, and CCL8 act via the receptor CCR2 (and possibly also CCR1) to stimulate migration of monocytes into the blood vessel wall (4).To suppress leukocyte recruitment in inflammatory diseases, numerous antagonists of specific chemokine receptors have been evaluated in clinical trials. However, these trials have not yielded any new antiinflammatory therapeutics (5), in part because most leukocytes can utilize multiple chemokine receptors, thus circumventing the specific antagonists. The alternative approach of targeting chemokines has not generally been favored, because it would require agents that bind with high affinity to multiple chemokines. However, the natural chemokine-binding proteins of ticks, helminths, and viruses (6–8) display multispecificity for mammalian chemokines, suggesting that they could potentially be deployed as antiinflammatory therapeutics.Evasins are two families of chemokine-binding, antiinflammatory proteins from tick saliva (6). Class A evasins each inhibit multiple CC chemokines of their mammalian hosts but none of the closely related CXC chemokines. Conversely, class B evasins are specific for CXC over CC chemokines but exhibit variable selectivity among CXC chemokines. Typically, each tick species secretes a mixture of evasins, thereby accomplishing broad-spectrum suppression of the host inflammatory response, presumably enabling the tick to feed on the host for extended periods.The in vivo antiinflammatory activity of tick evasins has been demonstrated using a variety of animal models of inflammatory diseases, including lung fibrosis, skin inflammation, arthritis, colitis, pancreatitis, ischemic reperfusion injury, postinfarction myocardial injury, and Leishmania major infection (9–13). However, deploying evasins as effective antiinflammatory therapeutics in humans would require engineering the natural evasins to selectively target the relevant array of chemokines for any given indication while minimizing off-target inhibition (14). Such engineering requires understanding both the specificity of evasins for a single chemokine subfamily and their target preference among chemokines within that subfamily.Previously, only a single structure has been reported for an evasin:chemokine complex, class A evasin EVA-1 bound to CCL3 (15), so it has not been possible to identify the conserved and variable features of the interactions. Nevertheless, the structure revealed that EVA-1 binds to several receptor recognition elements of CCL3, explaining its inhibitory activity. Moreover, limited mutational data (15, 16) have confirmed that residues in the N- and C-terminal regions of EVA-1 and the homologous EVA-4, respectively, contribute to binding affinity, raising the question of whether the structural basis of CC chemokine recognition varies across the class A evasin family.To establish a structure-based platform for engineering the chemokine selectivity of class A evasins, we now report the structures of EVA-P974 (previously called ACA-01) (17, 18), from the Cayenne tick (Amblyomma cajennense), bound to each of two wild-type chemokines and one chimeric CC chemokine. Structural comparisons and extensive evasin and chemokine mutational data revealed the structural basis for CC chemokine specificity and identified several “hotspots” that define target preference among CC chemokines. These insights enabled EVA-P974 to be engineered to modify its target preference. We further verified the molecular basis of the modified selectivity by solving the chemokine-bound structure of the engineered evasin. Finally, by inhibiting a chemokine mixture, we provide proof of principle for applying engineered evasins as multichemokine inhibitors.     

Ultrastructure of Clara cells stimulated by isoproterenol

The incidence of peripheral pulmonary adenocarcinoma has increased in recent years. Clara cell has been known as target for carcinogens and source of pulmonary tumors. One of the presumed roles of the bronchiolar Clara cell is the secretion of pulmonary surfactant into the bronchiolar lumen. To establish the secretory morphology of Clara cell, a well-defined secretory agonist, isoproterenol (500 mg/kg) and the antagonist, propranolol (20 mg/kg), were administered into five-week old mice. The secretory response was examined at 1 hour and 4 hours after injection. Ultrastructural morphometry was used to quantitate the secretory response by measuring area of apical cap of the Clara cells. Isoproterenol caused a significant increase in area of apical cap of Clara cells 1 and 4 hours after injection (p < 0.0001), while pretreatment with propranolol prevented this effect at 4 hours. Propranolol alone significantly decreased the area of Clara cells (p < 0.0001). Clara cells secretory granules disappeared 1 hour after propranolol plus isoproterenol administration, and the granules reappeared at 4 hours. The accelerated secretion of Clara cells by isoproterenol provides evidence of their secretory mechanism controlled by beta-adrenergic agonists. The study has confirmed the secretory role of Clara cells. The secretion is both apocrine and merocrine type.     

Meta-analysis of systemic sclerosis and exposure to solvents

BACKGROUND: Although systemic sclerosis is a rare disease, incidence rates have increased recently in the United States. This study investigated the association between systemic sclerosis and exposure to solvents. METHODS: A Medline search of articles published between 1966 and 2000 dealing with solvent exposure and systemic sclerosis identified eight studies that met inclusion criteria of the meta-analysis. The studies included seven case-control studies and one cohort study published between 1989 and 1998. A series of meta-analyses of studies on systemic sclerosis and solvent exposure were performed for all studies and for case-control studies. A random effect model was used to calculate a combined estimator of relative risk. RESULTS: The combined estimator of relative risk for all studies was 2.91, with a 95% confidence interval (CI) of 1.60 to 5.30. The combined estimator of relative risk for the seven case-control studies was 3.14 (95% CI, 1.56-6.33). CONCLUSIONS: A statistically significant increased relative risk for all studies and for case-control studies suggests that exposure to organic solvent may be a risk factor for developing systemic sclerosis. Further studies using other study designs and better control of confounders are needed.     

Mesenteric vein thrombosis in a patient heterozygous for factor Ⅴ Leiden and G20210A prothrombin genotypes

Mesenteric venous thrombosis(MVT)is a rare but life threatening form of bowel ischemia.It is implicated in 6%-9% of all cases of acute mesenteric ischemia.The proportion of patients with primary(or idiopathic)MVT varies from 0% to 49%,with a decrease in frequency secondary to more recent availability of newer investigations for hypercoagulability.The presence of factor Ⅴ Leiden(FVL)and prothrombin G20210A mutations(PGM)have been well documented in these cases.However,there have been scarce case reports describing MVT in heterozygotes of both these mutations occurring simultaneously and its implications on long term management.Our case describes acute MVT in a previously asymptomatic young patient with no prior history of venous thromboembolism.The patient was found to be heterozygous for FVL and PGM and treated with lifelong anticoagulation with warfarin(goal international normalized ratio:2-3)and avoidance of hormonal contraceptives.     

Concentration‐independent MRI of pH with a dendrimer‐based pH‐responsive nanoprobe

The measurement of extracellular pH (pH_e) has significant clinical value for pathological diagnoses and for monitoring the effects of pH‐altering therapies. One of the major problems of measuring pH_e with a relaxation‐based MRI contrast agent is that the longitudinal relaxivity depends on both pH and the concentration of the agent, requiring the use of a second pH‐unresponsive agent to measure the concentration. Here we tested the feasibility of measuring pH with a relaxation‐based dendritic MRI contrast agent in a concentration‐independent manner at clinically relevant field strengths. The transverse and longitudinal relaxation times in solutions of the contrast agent (GdDOTA‐4AmP)₄₄‐G5, a G5–PAMAM dendrimer‐based MRI contrast agent in water, were measured at 3 T and 7 T magnetic field strengths as a function of pH. At 3 T, longitudinal relaxivity (r₁) increased from 7.91 to 9.65 mM^?1 s^?1 (on a per Gd³⁺ basis) on changing pH from 8.84 to 6.35. At 7 T, r₁ relaxivity showed pH response, albeit at lower mean values; transverse relaxivity (r₂) remained independent of pH and magnetic field strengths. The longitudinal relaxivity of (GdDOTA‐4AmP)₄₄‐G5 exhibited a strong and reversible pH dependence. The ratio of relaxation rates R₂/R₁ also showed a linear relationship in a pH‐responsive manner, and this pH response was independent of the absolute concentration of (GdDOTA‐4AmP)₄₄‐G5 agent. Importantly, the nanoprobe (GdDOTA‐4AmP)₄₄‐G5 shows pH response in the range commonly found in the microenvironment of solid tumors. Copyright © 2015 John Wiley & Sons, Ltd.