A tick saliva serpin, IxsS17 inhibits host innate immune system proteases and enhances host colonization by Lyme disease agent.

Lyme disease (LD) caused by Borrelia burgdorferi is among the most important human vector borne diseases for which there is no effective prevention method. Identification of tick saliva transmission factors of the LD agent is needed before the highly advocated tick antigen-based vaccine could be developed. We previously reported the highly conserved Ixodes scapularis (Ixs) tick saliva serpin (S) 17 (IxsS17) was highly secreted by B. burgdorferi infected nymphs. Here, we show that IxsS17 promote tick feeding and enhances B. burgdorferi colonization of the host. We show that IxsS17 is not part of a redundant system, and its functional domain reactive center loop (RCL) is 100% conserved in all tick species. Yeast expressed recombinant (r) IxsS17 inhibits effector proteases of inflammation, blood clotting, and complement innate immune systems. Interestingly, differential precipitation analysis revealed novel functional insights that IxsS17 interacts with both effector proteases and regulatory protease inhibitors. For instance, rIxsS17 interacted with blood clotting proteases, fXII, fX, fXII, plasmin, and plasma kallikrein alongside blood clotting regulatory serpins (antithrombin III and heparin cofactor II). Similarly, rIxsS17 interacted with both complement system serine proteases, C1s, C2, and factor I and the regulatory serpin, plasma protease C1 inhibitor. Consistently, we validated that rIxsS17 dose dependently blocked deposition of the complement membrane attack complex via the lectin complement pathway and protected complement sensitive B. burgdorferi from complement-mediated killing. Likewise, co-inoculating C3H/HeN mice with rIxsS17 and B. burgdorferi significantly enhanced colonization of mouse heart and skin organs in a reverse dose dependent manner. Taken together, our data suggests an important role for IxsS17 in tick feeding and B. burgdorferi colonization of the host.

Association between focused cardiac ultrasound and time to furosemide administration in acute heart failure.

BACKGROUND: Heart failure (HF) is a global health burden, and its management in the emergency department (ED) is important. This study aimed to evaluate the association between focused cardiac ultrasound (FoCUS) and early administration of diuretics in patients with acute HF admitted to the ED. METHODS: This retrospective observational study was conducted at a tertiary academic hospital. Patients with acute HF patients who were admitted to the ED and receiving intravenous medication between January 2018 and December 2019 were enrolled. The main exposure was a FoCUS examination performed within 2 h of ED triage. The primary outcome was the time to furosemide administration. RESULTS: Of 1154 patients with acute HF, 787 were included in the study, with 116 of them having undergone FoCUS. The time to furosemide was significantly shorter in the FoCUS group (median time (q1-q3), 112 min; range, 65-163 min) compared to the non-FoCUS group (median time, 131 min; range, 71-229 min). In the multivariable logistic regression analysis adjusting for age, sex, chief complaint, mode of arrival, triage level, shock status, and desaturation at triage, early administration of furosemide within 2 h from triage was significantly higher in the FoCUS group (adjusted odds ratio, 1.63; 95% confidence intervals, 1.04-2.55) than in the non-FoCUS group. CONCLUSIONS: Early administration of intravenous furosemide was associated with FoCUS examination in patients with acute HF admitted to the ED. An early screening protocol could be useful for improving levels in clinical practice at EDs.

Analysis of the Common Femoral Artery and Vein: Anatomical Morphology, Vessel Relationship, and Factors Affecting Vessel Size.

Background and Objectives: We aimed to analyze the morphology of the common femoral artery (CFA) and common femoral vein (CFV) and the anatomical relationship between the two blood vessels, and to investigate the factors that influence the size of these blood vessels. Materials and Methods: This retrospective study included 584 patients who underwent abdominal and pelvic computed tomography from 1 February to 28 February 2021. We measured the vessels at three regions on both lower extremities (inguinal ligament, distal vessel bifurcation, midpoint) and analyzed and classified the degree of overlap between the CFA and CFV into three types, as well as the factors affecting vessel size. Results: After comparing the femoral vessels according to location, it was confirmed that the CFA and CFV were larger distally than proximally on both sides (p < 0.001). The degree of overlap increased distally (p < 0.001) but was less at the middle (p < 0.001) and distal (p = 0.011) regions on the right side. It was found that the size of CFA and CFV were related to age, sex, and body mass index (BMI) and that malignancy also affects the CFA size. Conclusions: The morphology of the CFA and CFV was conical and increased distally. The degree of overlap between the two blood vessels also increased distally but was less on the right than on the left. Age, sex, and BMI are significant factors affecting the sizes of the CFA and CFV, and malignancy is associated with the CFA size.

Borrelia burgdorferi infection modifies protein content in saliva of Ixodes scapularis nymphs.

BACKGROUND: Lyme disease (LD) caused by Borrelia burgdorferi is the most prevalent tick-borne disease. There is evidence that vaccines based on tick proteins that promote tick transmission of B. burgdorferi could prevent LD. As Ixodes scapularis nymph tick bites are responsible for most LD cases, this study sought to identify nymph tick saliva proteins associated with B. burgdorferi transmission using LC-MS/MS. Tick saliva was collected using a non-invasive method of stimulating ticks (uninfected and infected: unfed, and every 12 h during feeding through 72 h, and fully-fed) to salivate into 2% pilocarpine-PBS for protein identification using LC-MS/MS. RESULTS: We identified a combined 747 tick saliva proteins of uninfected and B. burgdorferi infected ticks that were classified into 25 functional categories: housekeeping-like (48%), unknown function (18%), protease inhibitors (9%), immune-related (6%), proteases (8%), extracellular matrix (7%), and small categories that account for <5% each. Notably, B. burgdorferi infected ticks secreted high number of saliva proteins (n=645) than uninfected ticks (n=376). Counter-intuitively, antimicrobial peptides, which function to block bacterial infection at tick feeding site were suppressed 23-85 folds in B. burgdorferi infected ticks. Similar to glycolysis enzymes being enhanced in mammalian cells exposed to B. burgdorferi : eight of the 10-glycolysis pathway enzymes were secreted at high abundance by B. burgdorferi infected ticks. Of significance, rabbits exposed to B. burgdorferi infected ticks acquired potent immunity that caused 40-60% mortality of B. burgdorferi infected ticks during the second infestation compared to 15-28% for the uninfected. This might be explained by ELISA data that show that high expression levels of immunogenic proteins in B. burgdorferi infected ticks. CONCLUSION: Data here suggest that B. burgdorferi infection modified protein content in tick saliva to promote its survival at the tick feeding site. For instance, enzymes; copper/zinc superoxide dismutase that led to production of H2O2 that is toxic to B. burgdorferi were suppressed, while, catalase and thioredoxin that neutralize H2O2, and pyruvate kinase which yields pyruvate that protects Bb from H2O2 killing were enhanced. We conclude data here is an important resource for discovery of effective antigens for a vaccine to prevent LD.

Amblyomma americanum ticks utilizes countervailing pro and anti-inflammatory proteins to evade host defense.

Feeding and transmission of tick-borne disease (TBD) agents by ticks are facilitated by tick saliva proteins (TSP). Thus, defining functional roles of TSPs in tick evasion is expected to reveal potential targets in tick-antigen based vaccines to prevent TBD infections. This study describes two types of Amblyomma americanum TSPs: those that are similar to LPS activate macrophage (MΦ) to express pro-inflammation (PI) markers and another set that suppresses PI marker expression by activated MΦ. We show that similar to LPS, three recombinant (r) A. americanum insulin-like growth factor binding-related proteins (rAamIGFBP-rP1, rAamIGFBP-rP6S, and rAamIGFBP-rP6L), hereafter designated as PI-rTSPs, stimulated both PBMC -derived MΦ and mice RAW 267.4 MΦ to express PI co-stimulatory markers, CD40, CD80, and CD86 and cytokines, TNFα, IL-1, and IL-6. In contrast, two A. americanum tick saliva serine protease inhibitors (serpins), AAS27 and AAS41, hereafter designated as anti-inflammatory (AI) rTSPs, on their own did not affect MΦ function or suppress expression of PI markers, but enhanced expression of AI cytokines (IL-10 and TGFß) in MΦ that were pre-activated by LPS or PI-rTSPs. Mice paw edema test demonstrated that in vitro validated PI- and AI-rTSPs are functional in vivo since injection of HEK293-expressed PI-rTSPs (individually or as a cocktail) induced edema comparable to carrageenan-induced edema and was characterized by upregulation of CD40, CD80, CD86, TNF-α, IL-1, IL-6, and chemokines: CXCL1, CCL2, CCL3, CCL5, and CCL11, whereas the AI-rTSPs (individually and cocktail) were suppressive. We propose that the tick may utilize countervailing PI and AI TSPs to regulate evasion of host immune defenses whereby TSPs such as rAamIGFBP-rPs activate host immune cells and proteins such as AAS27 and AAS41 suppress the activated immune cells.

A Mobile Videoconference-Based Intervention on Stress Reduction and Resilience Enhancement in Employees: Randomized Controlled Trial.

BACKGROUND: Videoconferencing-based treatments have shown great potential in increasing engagement and compliance by decreasing the barriers of time and distance. In general, employees tend to experience a lot of stress, but find it difficult to visit a clinic during office hours. OBJECTIVE: The purpose of this study was to investigate the effectiveness of a mobile videoconference-based intervention for stress reduction and resilience enhancement in employees. METHODS: In total, 81 participants were randomly allocated to one of the three conditions: mobile videoconferencing, in-person, and self-care; of these, 72 completed the study. All participants underwent assessment via self-reported questionnaires before, immediately after, and 1 month after the intervention. Intervention lasted for 4 weeks and consisted of elements of cognitive behavioral therapy, positive psychology, and meditation. Changes in clinical variables regarding stress and resilience across time were compared between treatment conditions. RESULTS: There were significant condition × time effects on variables measuring perceived stress, resilience, emotional labor, and sleep, demonstrating significantly differential effects across time according to treatment condition. Moreover, there were significant effects of condition on perceived stress and occupational stress. There were no significant differences in any variable between the mobile videoconferencing and in-person conditions at 1 month after the intervention. CONCLUSIONS: Results indicate that both mobile videoconferencing and in-person interventions were comparably effective in decreasing stress and enhancing resilience. Further studies with a larger sample size and a longer follow-up period are warranted to investigate the long-term effect of mobile videoconferencing interventions. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT03256682; https://clinicaltrials.gov/ct2/show/NCT03256682 (Archived by WebCite at http://www.webcitation.org/71W77bwnR).

Synovial osteochondromatosis in the subacromial bursa mimicking calcific tendinitis: sonographic diagnosis.

Synovial osteochondromatosis is an idiopathic benign metaplasia of the synovial membrane rarely found in an extra-articular bursa. We describe the case of a 55-year-old woman with synovial osteochondromatosis in the subacromial bursa mimicking calcific tendinitis. Plain radiographs showed a radiopaque mass over the middle facet of the greater tuberosity, suggesting calcific tendinitis. Sonography, however, showed a loose body in the subacromial bursa, and no evidence of calcification inside the rotator cuff.

Bioinformatics and expression analyses of the Ixodes scapularis tick cystatin family.

The cystatins are inhibitors of papain- and legumain-like cysteine proteinases, classified in MEROPS subfamilies I25A-I25C. This study shows that 84 % (42/50) of tick cystatins are putatively extracellular in subfamily I25B and the rest are putatively intracellular in subfamily I25A. On the neighbor joining phylogeny guide tree, subfamily I25A members cluster together, while subfamily I25B cystatins segregate among prostriata or metastriata ticks. Two Ixodes scapularis cystatins, AAY66864 and ISCW011771 that show 50-71 % amino acid identity to metastriata tick cystatins may be linked to pathways that are common to all ticks, while ISCW000447 100 % conserved in I. ricinus is important among prostriata ticks. Likewise metastriata tick cystatins, Dermacentor variabilis-ACF35512, Rhipicephalus microplus-ACX53850, A. americanum-AEO36092, R. sanguineus-ACX53922, D. variabilis-ACF35514, R. sanguineus-ACX54033 and A. maculatum-AEO35155 that show 73-86 % amino acid identity may be essential to metastriata tick physiology. RT-PCR expression analyses revealed that I. scapularis cystatins were constitutively expressed in the salivary glands, midguts and other tissues of unfed ticks and ticks that were fed for 24-120 h, except for ISCW017861 that are restricted to the 24 h feeding time point. On the basis of mRNA expression patterns, I. scapularis cystatins, ISCW017861, ISCW011771, ISCW002215 and ISCW0024528 that are highly expressed at 24 h are likely involved in regulating early stage tick feeding events such as tick attachment onto host skin and creation of the feeding lesion. Similarly, ISCW018602, ISCW018603 and ISCW000447 that show 2-3 fold transcript increase by 120 h of feeding are likely associated with blood meal up take, while those that maintain steady state expression levels (ISCW018600, ISCW018601 and ISCW018604) during feeding may not be associated with tick feeding regulation. We discuss our findings in the context of advancing our knowledge of tick molecular biology.

Stable internal reference genes for quantitative RT-PCR analyses in Rhipicephalus microplus during embryogenesis.

Studies on the transcriptional control of gene expression are crucial to understand changes in organism's physiological or cellular conditions. To obtain reliable data on mRNA amounts and the estimation of gene expression levels, it is crucial to normalize the target gene with one or more internal reference gene(s). However, the use of constitutive genes as reference genes is controversial, as their expression patterns are sometimes more complex than previously thought. In various arthropod vectors, including ticks, several constitutive genes have been identified by studying gene expression in different tissues and life stages. The cattle tick Rhipicephalus microplus is a major vector for several pathogens and is widely distributed in tropical and subtropical regions globally. Tick developmental physiology is an essential aspect of research, particularly embryogenesis, where many important developmental events occur, thus the identification of stable reference genes is essential for the interpretation of reliable gene expression data. This study aimed to identify and select R. microplus housekeeping genes and evaluate their stability during embryogenesis. Reference genes used as internal control in molecular assays were selected based on previous studies. These genes were screened by quantitative PCR (qPCR) and tested for gene expression stability during embryogenesis. Results demonstrated that the relative stability of reference genes varied at different time points during the embryogenesis. The GeNorm tool showed that elongation factor 1α (Elf1a) and ribosomal protein L4 (Rpl4) were the most stable genes, while H3 histone family 3A (Hist3A) and ribosomal protein S18 (RpS18) were the least stable. The NormFinder tool showed that Rpl4 was the most stable gene, while the ranking of Elf1a was intermediate in all tested conditions. The BestKeeper tool showed that Rpl4 and cyclophilin A (CycA) were the more and less stable genes, respectively. These data collectively demonstrate that Rpl4, Elf1a, and GAPDH are suitable internal controls for normalizing qPCR during R. microplus embryogenesis. These genes were consistently identified as the most stable in various analysis methods employed in this study. Thus, findings presented in this study offer valuable information for the study of gene expression during embryogenesis in R. microplus.

Ixodes scapularis nymph saliva protein blocks host inflammation and complement-mediated killing of Lyme disease agent, Borrelia burgdorferi.

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent.

Identification and characterization of proteins that form the inner core Ixodes scapularis tick attachment cement layer.

Ixodes scapularis long-term blood feeding behavior is facilitated by a tick secreted bio adhesive (tick cement) that attaches tick mouthparts to skin tissue and prevents the host from dislodging the attached tick. Understanding tick cement formation is highly sought after as its disruption will prevent tick feeding. This study describes proteins that form the inner core layer of I. scapularis tick cement as disrupting these proteins will likely stop formation of the outer cortical layer. The inner core cement layer completes formation by 24 h of tick attachment. Thus, we used laser-capture microdissection to isolate cement from cryosections of 6 h and 24 h tick attachment sites and to distinguish between early and late inner core cement proteins. LC-MS/MS analysis identified 138 tick cement proteins (TCPs) of which 37 and 35 were unique in cement of 6 and 24 h attached ticks respectively. We grouped TCPs in 14 functional categories: cuticular protein (16%), tick specific proteins of unknown function, cytoskeletal proteins, and enzymes (13% each), enzymes (10%), antioxidant, glycine rich, scaffolding, heat shock, histone, histamine binding, proteases and protease inhibitors, and miscellaneous (3-6% each). Gene ontology analysis confirm that TCPs are enriched for bio adhesive properties. Our data offer insights into tick cement bonding patterns and set the foundation for understanding the molecular basis of I. scapularis tick cement formation.

Amblyomma americanum (L.) (Acari: Ixodidae) tick salivary gland serine protease inhibitor (serpin) 6 is secreted into tick saliva during tick feeding.

In order to successfully feed and transmit disease agents, ticks are thought to inject serine protease inhibitors (serpins) into the host to modulate host defense responses to tick feeding, such as inflammation, the complement activation pathway and blood coagulation. In this study, we show that Amblyomma americanum (Aam) serpin (S) 6 is putatively injected into the host during tick feeding, in that the antibody to recombinant (r) AamS6 specifically reacted with the expected ∼43/45 kDa AamS6 protein band on western blots of pilocarpine-induced tick saliva. Additionally, antibodies to tick saliva proteins that were generated by repeated 48 h infestations of rabbits with adult A. americanum specifically reacted with rAamS6. We speculate that AamS6 is associated with regulating events at the start of the tick feeding process, as temporal and spatial RT-PCR and western blot analyses revealed that both AamS6 mRNA and protein are strongly expressed during the first 24-72 h of feeding time before starting to fade from 96 h. The AamS6 protein has an apparently slow turnover rate in that, although the injection of AamS6 dsRNA into unfed ticks triggered complete disruption of the AamS6 mRNA by the 48 h feeding time point, western blot analysis of protein extracts of the same animals showed that the AamS6 protein that may have been expressed prior to disruption of the AamS6 mRNA was not depleted. We speculate that the presence of the AamS6 protein in ticks despite the complete disruption of the AamS6 mRNA explains the observation that RNAi-mediated silencing of the AamS6 mRNA did not affect the ability of A. americanum ticks to attach onto host skin, successfully feed and lay eggs. These findings are discussed in regards to advances in the molecular biology of ticks.

Shed blood re-transfusion provides no benefit in computer-assisted primary total knee arthroplasty.

PURPOSE: This matched case-cohort retrospective study examined the effectiveness of shed blood re-transfusion in reducing the need for allogeneic blood transfusion in computer-assisted primary cemented total knee arthroplasty (TKA). METHODS: The shed blood re-transfusion system used was the cell saver system. Data from 146 cases were analyzed (73 patients with cell saver, 73 patients without cell saver). RESULTS: The ABT rate was similar in each group. The mean allogenic blood transfusion volume was similar for each group (CS=214±453 ml, non-CS=288±447 ml). The only factors correlated with allogenic blood transfusion use were low preoperative hemoglobin and low body mass index. Two patients in cell saver group experienced shivering after re-transfusion. CONCLUSION: Shed blood re-transfusion provided no blood management benefits in computer-assisted primary TKA and is therefore recommended only for selected patients with low hemoglobin levels and low body mass index.

Amblyomma americanum serpin 41 (AAS41) inhibits inflammation by targeting chymase and chymotrypsin.

Ticks inject serine protease inhibitors (serpins) into their feeding sites to evade serine protease-mediated host defenses against tick-feeding. This study describes two highly identitical (97%) but functionally different Amblyomma americanum tick saliva serpins (AAS41 and 46) that are secreted at the inception of tick-feeding. We show that AAS41, which encodes a leucine at the P1 site inhibits inflammation system proteases: chymase (SI = 3.23, Ka = 5.6 ± 3.7X103M-1 s-1) and α-chymotrypsin (SI = 3.18, Ka = 1.6 ± 4.1X104M-1 s-1), while AAS46, which encodes threonine has no inhibitory activity. Similary, rAAS41 inhibits rMCP-1 purified from rat peritonuem derived mast cells. Consistently, rAAS41 inhibits chymase-mediated inflammation induced by compound 48/80 in rat paw edema and vascular permeability models. Native AAS41/46 proteins are among tick saliva immunogens that provoke anti-tick immunity in repeatedly infested animals as revealed by specific reactivity with tick immune sera. Of significance, native AAS41/46 play critical tick-feeding functions in that RNAi-mediated silencing caused ticks to ingest significantly less blood. Importantly, monospecific antibodies to rAAS41 blocked inhibitory functions of rAAS41, suggesting potential for design of vaccine antigens that provokes immunity to neutralize functions of this protein at the tick-feeding site. We discuss our findings with reference to tick-feeding physiology and discovery of effective tick vaccine antigens.

Time-resolved proteomic profile of Amblyomma americanum tick saliva during feeding.

Amblyomma americanum ticks transmit more than a third of human tick-borne disease (TBD) agents in the United States. Tick saliva proteins are critical to success of ticks as vectors of TBD agents, and thus might serve as targets in tick antigen-based vaccines to prevent TBD infections. We describe a systems biology approach to identify, by LC-MS/MS, saliva proteins (tick = 1182, rabbit = 335) that A. americanum ticks likely inject into the host every 24 h during the first 8 days of feeding, and towards the end of feeding. Searching against entries in GenBank grouped tick and rabbit proteins into 27 and 25 functional categories. Aside from housekeeping-like proteins, majority of tick saliva proteins belong to the tick-specific (no homology to non-tick organisms: 32%), protease inhibitors (13%), proteases (8%), glycine-rich proteins (6%) and lipocalins (4%) categories. Global secretion dynamics analysis suggests that majority (74%) of proteins in this study are associated with regulating initial tick feeding functions and transmission of pathogens as they are secreted within 24-48 h of tick attachment. Comparative analysis of the A. americanum tick saliva proteome to five other tick saliva proteomes identified 284 conserved tick saliva proteins: we speculate that these regulate critical tick feeding functions and might serve as tick vaccine antigens. We discuss our findings in the context of understanding A. americanum tick feeding physiology as a means through which we can find effective targets for a vaccine against tick feeding.

Amblyomma americanum serpin 27 (AAS27) is a tick salivary anti-inflammatory protein secreted into the host during feeding.

Ticks successfully feed and transmit pathogens by injecting pharmacological compounds in saliva to thwart host defenses. We have previously used LC-MS/MS to identify proteins that are present in saliva of unfed Amblyomma americanum ticks that were exposed to different hosts. Here we show that A. americanum serine protease inhibitor (serpin) 27 (AAS27) is an immunogenic saliva protein that is injected into the host within the first day of tick feeding and is an anti-inflammatory protein that might act by blocking plasmin and trypsin functions. Although AAS27 is injected into the host throughout tick feeding, qRT-PCR and western blotting analyses indicate that the respective transcript and protein are present in high amounts within the first 24 h of tick feeding. Biochemical screening of Pichia pastoris-expressed recombinant (r) AAS27 against mammalian proteases related to host defense shows it is an inhibitor of trypsin and plasmin, with stoichiometry of inhibition indices of 3.5 and 3.8, respectively. Consistent with typical inhibitory serpins, rAAS27 formed heat- and SDS-stable irreversible complexes with both proteases. We further demonstrate that rAAS27 inhibits trypsin with ka of 6.46 ± 1.24 x 104 M-1 s-1, comparable to serpins of other tick species. We show that native AAS27 is part of the repertoire of proteins responsible for the inhibitory activity against trypsin in crude tick saliva. AAS27 is likely utilized by the tick to evade the hosts inflammation defense since rAAS27 blocks both formalin and compound 48/80-induced inflammation in rats. Tick immune sera of rabbits that had acquired resistance against tick feeding following repeated infestations with A. americanum or Ixodes scapularis ticks reacts with rAAS27. Of significant interest, antibody to rAAS27 blocks this serpin inhibitory functions. Taken together, we conclude that AAS27 is an anti-inflammatory protein secreted into the host during feeding and may represent a potential candidate for development of an anti-tick vaccine.

Post-steroid panniculitis in an adult.

Post-steroid panniculitis is known to be very rare and most of the reported cases have been in children after corticosteroid therapy. We present a case of post-steroid panniculitis occurring in a 60-year-old man after massive, long-term administration of corticosteroids for acute exacerbation of chronic obstructive pulmonary disease (COPD). Histopathological examination of a nodule revealed a patchy area of fat necrosis, several multinucleated giant cells containing needle-shaped clefts. The lesions subsided completely in approximately 12 weeks without any treatment. We suggest that post-steroid panniculitis is not confined to childhood but also occurs in adulthood.

Disruption of blood meal-responsive serpins prevents Ixodes scapularis from feeding to repletion.

Serine protease inhibitors (serpins) are thought to mediate the tick's evasion of the host's serine protease-mediated defense pathways such as inflammation and blood clotting. This study describes characterization and target validation of 11 blood meal-responsive serpins that are associated with nymph and adult Ixodes scapularis tick feeding as revealed by quantitative (q)RT-PCR and RNAi silencing analyses. Given the high number of targets, we used combinatorial (co) RNAi silencing to disrupt candidate serpins in two groups (G): seven highly identical and four non-identical serpins based on amino acid identities, here after called GI and GII respectively. We show that injection of both GI and GII co-dsRNA into unfed nymph and adult I. scapularis ticks triggered suppression of cognate serpin mRNA. We show that disruption of GII, but not GI serpins significantly reduced feeding efficiency of both nymph and adult I. scapularis ticks. Knockdown of GII serpin transcripts caused significant respective mortalities of ≤40 and 71% of nymphal and adult ticks that occurred within 24-48 h of attachment. This is significant, as the observed lethality preceded the tick feeding period when transmission of tick borne pathogens is predominant. We suspect that some of the GII serpins (S9, S17, S19 and S32) play roles in the tick detachment process in that upon detachment, mouthparts of GII co-dsRNA injected were covered with a whitish gel-like tissue that could be the tick cement cone. Normally, ticks do not retain tissue on their mouthparts upon detachment. Furthermore, disruption of GII serpins reduced tick blood meal sizes and the adult tick's ability to convert the blood meal to eggs. We discuss our data with reference to tick feeding physiology and conclude that some of the GII serpins are potential targets for anti-tick vaccine development.

Identification and characterization of proteins in the Amblyomma americanum tick cement cone.

The adaptation of hard ticks to feed for long periods is facilitated by the cement cone, which securely anchors the tick mouthparts onto host skin and protects the tick from being groomed off by the host. Thus, preventing tick cement deposition is an attractive target for the development of innovative tick control. We used LC-MS/MS sequencing to identify 160 Amblyomma americanum tick cement proteins that include glycine-rich proteins (GRP, 19%), protease inhibitors (12%), proteins of unknown function (11%), mucin (4%), detoxification, storage, and lipocalin at 1% each, and housekeeping proteins (50%). Spatiotemporal transcription analysis showing mRNA expression in multiple tick organs and transcript abundance increasing with feeding suggest that selected GRPs (n = 13) regulate multiple tick feeding functions, being classified as constitutively expressed (CE), feeding induced (FI), and up-regulated with feeding (UR). We show that transcription of CE GRPs is likely under the control of tick appetence associated factors in that mRNA abundance increased several thousand fold in 1 week old adult ticks, the time period that coincides with tick attainment of appetence. Given the high number of targets, we synthesized and injected unfed ticks with combinatorial (co) double stranded (ds)RNA and disrupted GRP mRNA in clusters according to similar transcription patterns: CE (n = 3), FI, (n = 4), and UR (n = 6) to streamline the work. Our data suggest that CE and FI GRPs are important for maintenance of the tick feeding site in that reddening and subsequent bleeding were observed around the mouthparts of CE and FI GRP co-dsRNA injected ticks during feeding. Furthermore, although not significantly different, indices for blood meal size and fecundity were apparently reduced in FI and UR ticks. We discuss our data with reference to A. americanum tick feeding physiology.