Site-directed mutagenesis of the CC chemokine binding protein 35K-Fc reveals residues essential for activity and mutations that increase the potency of CC chemokine blockade

Chemokines of the CC class are key mediators of monocyte recruitment and macrophage differentiation and have a well documented role in many inflammatory diseases. Blockade of chemokine activity is therefore an attractive target for anti-inflammatory therapy. 35K (vCCI) is a high-affinity chemokine binding protein expressed by poxviruses, which binds all human and murine CC chemokines, preventing their interaction with chemokine receptors. We developed an Fc-fusion protein of 35K with a modified human IgG1 Fc domain and expressed this construct in human embryonic kidney 293T cells. Purified 35K-Fc is capable of inhibiting CC chemokine-induced calcium flux, chemotaxis, and β-arrestin recruitment in primary macrophages and transfected cells. To elucidate the residues involved in chemokine neutralization, we performed site-directed mutagenesis of six key amino acids in 35K and expressed the mutant Fc-fusion proteins in vitro. We screened the mutants for their ability to block chemokine-induced β-arrestin recruitment in transfected cells and to inhibit primary macrophage signaling in an electric cell substrate impedance sensing assay. Using a sterile model of acute inflammation, zymosan-induced peritonitis, we confirmed that wild-type 35K-Fc can reduce monocyte recruitment, whereas one mutant (R89A) showed a more pronounced blockade of monocyte influx and another mutant (E143K) showed total loss of function. We believe that 35K-Fc will be a useful tool for exploring the role of CC chemokines in chronic inflammatory pathologies, and we have identified a higher potency form of the molecule that may have potential therapeutic applications in chronic inflammatory disease.     

Effects of Warm-Up on Vertical Jump Performance and Muscle Electrical Activity Using Half-Squats at Low and Moderate Intensity

The purpose of this study was to determine the effects of a specific warm-up using half-squats at low and moderate intensity on vertical jump performance and electromyographic activity of the thigh muscles. The subjects were 26 men who were divided into a low intensity group (LIG; n = 13) and a moderate intensity group (MIG; n = 13). The LIG performed a specific warm-up protocol that included the explosive execution of half-squats with loads 25 and 35% of the one repetition maximum (1RM) and the MIG with loads 45 and 65% of the 1RM. The two groups performed a countermovement jump (CMJ) before and three minutes after the specific warm-up protocols. During the concentric phase of the CMJ a linear encoder connected to an A/D converter interfaced to a PC with a software for data acquisition and analysis allowed the calculation of average mechanical power. The electromyographic (EMG) activity of the vastus lateralis (VL), vastus medialis (VM) and rectus femoris (RF) were recorded during the concentric phase of the jumps. The average quadriceps (Qc) activity (mean value of the VL, VM and RF) was also calculated. A two way ANOVA (protocols X time) with repeated measures on the second factor was used to analyze the data. Following the specific warm-up procedure both groups improved (p ≤ 0.05) CMJ performance and mechanical power by 3.5% and 6.3%, respectively, with no differences observed between the two groups. EMG activity of the Qc and VL increased (p ≤ 0.05) for both groups by 5.9% and 8.5%, respectively. It is concluded that the use of a specific warm-up that includes half-squats, performed explosively with low to moderate intensity, improves CMJ performance. This may be due to increased muscle activation as evaluated by the surface EMG.

Key points

• The inclusion of two sets of explosively performed half squats with low to moderate loads in the warm up procedure elicited an acute performance en-hancement.
• The performance was enhanced regardless of the load used in the warm-up.
• The performance enhancement is accompanied by a greater electromyographic activity of the knee extensors muscles.

Key words: EMG, contrast training, resistance exercise     

A prospective study of infantile hemangiomas with a focus on incidence and risk factors

Infantile hemangiomas (IHs) are the most common tumor of infancy and have been estimated to occur in 4% of infants. Only two previous incidence studies of IH in a healthy population have been published, and both of these were performed in the first week of life. The objective was to identify the incidence of IH in an Australian neonatal population and characterize the risk factors. All women who presented to the postnatal ward in a 200-bed maternity hospital were asked to complete a questionnaire. Details of maternal history and birth details were recorded. Two follow-up emails 3 and 6 weeks after discharge were sent to all mothers who consented, asking if their baby had developed an IH. Babies reported to have an IH were seen in clinic to confirm the diagnosis. Details were collected from 1,034 mothers of 1,065 babies; 28 (2.6%) of the infants developed IH. Babies that developed IH were more likely to be female (p < 0.001), have a low birth weight (p = 0.020), be born at a gestational age of <37 weeks (p = 0.005), and be conceived through in vitro fertilization (IVF) (p = 0.001) than those who did not. The incidence of IH at 6 weeks of life was 2.6%.     

The effects of body weight status on orthostatic intolerance and predisposition to noncardiac syncope

Orthostatic intolerance (OI) is frequently the mechanism underlying the occurrence of noncardiac syncope (NCS) and is associated with substantial risk for injury. Body weight status appears to be a modifier of orthostatic responses and possibly influences the propensity to NCS. The majority of cross‐sectional studies have found that the lower the body mass index (BMI) the greater the predisposition to OI is, accompanied with both down‐regulation of sympathetic nervous system activity and up‐regulation of parasympathetic nervous system activity. These changes appear to occur across the whole spectrum of BMI values from underweight to obesity, while they may be associated more strongly with central body fat than total body fat. Weight loss following bariatric surgery has been consistently found to increase OI, attributed first to the effects of weight loss per se, second to the specific type of surgical procedure and third to the potential postoperative autonomic neuropathy due to vitamin deficiency. The increased OI following bariatric surgery renders this intervention not easily tolerable for the affected individuals, mandating increased fluid and salt intake, pharmacological measures or surgical adjustments to attenuate OI. All future studies investigating orthostatic responses and NCS should implement a matching of the population arms for BMI and ideally for body fat.     

Children achieve adult-like sensory integration during stance at 12-years-old

This study examined balance control in children to determine at what age the integration of sensory information in unperturbed stance is comparable to that of adults. In addition, it examined whether overall performance was related to age, gender and specific physical characteristics, such as height, weight and body mass index (BMI). Seventy-four female and 80 male children between the ages of 6 and 12 years participated in the study, as well as 20 adults, aged 20–22 years. The Sensory Organization Test (SOT), a component of computerized dynamic posturography, was used to assess overall balance as well as the use of specific sensory information in maintaining stability. Analyses of variance revealed significant differences between equilibrium scores of 7- and 8-year-old and 11- and 12-year-old (p < .01), with only the 12-year-old participants achieving scores comparable to those of the adult group. A repeated-measures analysis of variance comparing the use of different sensory information across age and gender groups revealed that while all groups demonstrated mature use of somatosensory information, children under 11 did not use visual information as effectively as adults, and only the 12-year-old group demonstrated adult-like use of vestibular information. Correlational analyses revealed a moderate correlation between composite balance scores and age, r(152) = .38, p < .001, but poor correlations between the composite equilibrium score and height, weight and BMI (r < .13, p > .15). Multiple regression analysis revealed that while physical characteristics accounted for approximately 20% of the variability in the composite equilibrium score, age alone accounted for the largest single contribution to the variance (16%). These results support recent findings suggesting that children do not demonstrate adult-like use of sensory information prior to age 12 years.     

Effect of fluoride dentifrices on white spot lesions during orthodontic treatment: A randomized trial

Objectives:To determine the effect of Clinpro 5000, Clinpro Tooth Crème, and MI-Paste Plus on the formation of white spot lesions in patients undergoing orthodontic treatment.Materials and Methods:Three prospective groups with 40 patients undergoing orthodontic treatment in each group were evaluated (total recruitment = 120 subjects). The selected product was brushed on for 2 minutes twice daily for 4 months. Subjects were reviewed for 4 months on a monthly basis. The Enamel Decalcification Index (EDI) was used to determine the number of white spot lesions per surface at each visit.Results:100 subjects (35 using Clinpro 5000, 32 using Clinpro Tooth Crème, and 33 using MI Paste Plus) completed the study. The data lend strong support for Clinpro 5000 providing superior protection against enamel decalcification when compared to Clinpro Crème, and mixed support when compared to MI Paste Plus.Conclusions:The use of Clinpro 5000, Clinpro Crème, and MI paste Plus all have a reduction effect on white spot lesions when compared to studies reported previously. Clinpro 5000 has a marginally better effect than the two other test pastes. The results of this study can be used by clinicians when deciding the effectiveness of using fluoride dentifrice products to prevent white spot lesions in their orthodontic practice (ClinicalTrials.gov ID: {"type":"clinical-trial","attrs":{"text":"NCT03440996","term_id":"NCT03440996"}}NCT03440996).     

Proliferative nodules of undifferentiated spindle cells arising in a large congenital melanocytic naevus

Proliferative nodules (PN) are benign lesions that arise in large congenital melanocytic naevi (LCMN). Clinically and histologically they can be difficult to differentiate from malignancies, which are also associated with LCMN. The PN in this case consisted of undifferentiated spindle cells and exhibited unusual histological features including negative stains for melanocytic markers (S100, HMB45 and MelA), negative stain for c‐Kit, high mitotic index and unusual morphology of the lesional cells. As a result, a firm histological classification could not be made, which posed a challenge for the clinical management.     

Lack of cytokine-induced skin reaction correlates with acute physiology score and mortality in patients receiving intensive care

Because the majority of patients receiving intensive care are anergic to ubiquitous skin test antigens, prediction of clinical outcome cannot be based on their delayed-type hypersensitivity score, which is 0, but which is also a strong indicator of sepsis-related mortality in the preoperative patient. However, a cytokine-rich supernatant generated in the mixed lymphocyte culture reaction is able to induce an early skin reaction that peaks at 12 hours and wanes by 24 hours in some, but not all, anergic patients. Assessment of the clinical course of these patients demonstrated that only three of 25 patients who had a skin reaction larger than 5 mm died, compared with ten of 16 patients who failed to display this early reaction. Further analysis showed that both the cytokine-induced reaction and the acute physiology score significantly and independently contributed to the probability of death in anergic patients. These data suggest that the ability to generate a local inflammatory response to cytokines in anergic patients identifies a subpopulation of patients who have maintained some host-defense responsiveness and this response can be utilized to predict outcome.     

Immunomodulation by altered gastrointestinal tract flora. The effects of orally administered, killed Staphylococcus epidermidis, Candida, and Pseudomonas on systemic immune responses

We investigated the role of microbial products from the gastrointestinal tract in the pathogenesis of immunosuppression in critical illness. Rats were gavaged daily for three weeks with killed Staphylococcus epidermidis, Candida, and Pseudomonas--organisms frequently isolated from the upper gastrointestinal tract of the critically ill patient; nonspecific immunity was measured by the delayed-type hypersensitivity (DTH) response. Gavage with either Pseudomonas or Candida resulted in significant suppression of DTH responses, while gavage with S epidermidis produced modest enhancement of DTH. Animals given Pseudomonas demonstrated impaired ability to localize a subcutaneous Staphylococcus aureus challenge and decreased IgM but enhanced anti-Pseudomonas IgA and IgG responses after intraperitoneal immunization. Pulmonary bacterial clearance at seven days was normal. These findings suggest that gut colonization with Candida or Pseudomonas may contribute to impairment of cell-mediated immunity in the critically ill patient.     

Altered leukocyte delivery to specific and nonspecific inflammatory skin lesions following burn injury

This study assessed the effect of burn trauma on the in vivo leukocyte cell delivery during the first 24 hr of the delayed type hypersensitivity (DTH) skin test reaction and a bacterial skin abscess. Inbred male Lewis rats sensitized to keyhole limpet hemocyanin (KLH) were given a 30% scald burn or sham burn. Three days later the animals were injected intradermally, at different sites, with 0.3 mg of KLH, 10(8) organisms of S. aureus 502A, and 0.1 cc of saline, at 2 to 24 hr. Leukocytes labelled with Indium111 oxine(leu111) were injected intravenously. In sham rats the peak leu111 influx in the DTH reaction occurred at 2-4 hr while in the abscess it was biphasic with peaks at 3 hr and 6-8 hr. In burn trauma rats there was a markedly increased leu111 peak at 2 hr in both the DTH and abscess reactions followed by a significantly lower than normal leu111 delivery in the late (6-24) hours. This marked early leukocyte influx in burned rats was paralleled by a reduced DTH skin test lesion (8.2 +/- 1.1 mm to 4.2 +/- 1.1 mm) and an increased bacterial abscess (5.1 +/- 1.1 mm to 8.1 +/- 0.9 mm) post burn. There was a direct correlation between leukocyte cell delivery to a DTH reaction and a bacterial abscess (r8 = 0.69, Spearman rank; p less than 0.001). We conclude that burn trauma results in altered leukocyte delivery to inflammatory lesions and the DTH response can be used to assess the ability of a burn trauma host to recruit leukocytes at a site of infection.