R. Alexander Blackwood

Clustering of Urokinase Receptors (uPAR; CD87) Induces Proinflammatory Signaling in Human Polymorphonuclear Neutrophils

Leukocytes use urokinase receptors (uPAR; CD87) in adhesion, migration, and proteolysis of matrix proteins. Typically, uPAR clusters at cell-substratum interfaces, at focal adhesions, and at the leading edges of migrating cells. This study was undertaken to determine whether uPAR clustering mediates activation signaling in human polymorphonuclear neutrophils. Cells were labeled with fluo-3/AM to quantitate intracellular Ca2+ ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by Ab cross-linking. Aggregating uPAR induced a highly reproducible increase in [Ca2+]i (baseline to peak) of 295 ± 37 nM (p = 0.0002). Acutely treating cells with high m.w. urokinase (HMW-uPA; 4000 IU/ml) produced a response of similar magnitude but far shorter duration. Selectively aggregating uPA-occupied uPAR produced smaller increases in [Ca2+]i, but saturating uPAR with HMW-uPA increased the response to approximate that of uPAR cross-linking. Cross-linking uPAR induced rapid and significant increases in membrane expression of CD11b and increased degranulation (release of β-glucuronidase and lactoferrin) to a significantly greater degree than cross-linking control Abs. The magnitude of degranulation correlated closely with the difference between baseline and peak [Ca2+]i, but was not dependent on the state of uPA occupancy. By contrast, selectively cross-linking uPA-occupied uPAR was capable of directly inducing superoxide release as well as enhancing FMLP-stimulated superoxide release. These results could not be duplicated by preferentially cross-linking unoccupied uPAR. We conclude that uPAR aggregation initiates activation signaling in polymorphonuclear neutrophils through at least two distinct uPA-dependent and uPA-independent pathways, increasing their proinflammatory potency (degranulation and oxidant release) and altering expression of CD11b/CD18 to favor a firmly adherent phenotype.

Cutting edge: Evidence for a signaling partnership between urokinase receptors (CD87) and L-selectin (CD62L) in human polymorphonuclear neutrophils

Leukocyte urokinase plasminogen activator receptors (uPARs) cluster at adhesion interfaces and at migratory fronts where they participate in adhesion, chemotaxis, and proteolysis. uPAR aggregation triggers activation signaling even though this glycolipid-anchored protein must associate with membrane-spanning proteins to access the cell interior. This study demonstrates a novel partnership between uPAR and L-selectin in human polymorphonuclear neutrophils. Fluorescence resonance energy transfer demonstrated a direct physical association between uPAR and L-selectin. To examine the role of L-selectin in uPAR-mediated signaling, uPAR was cross-linked and intracellular Ca2+ concentrations were measured by spectrofluorometry. A mAb reactive against the carbohydrate binding domain (CBD) of L-selectin substantially inhibited uPAR-mediated Ca2+ mobilization, whereas mAbs against the β2 integrin complement receptor 3 (CR3), another uPAR-binding adhesion protein, had no effect. Similarly, fucoidan, a sulfated polysaccharide that binds to L-selectin CBD, inhibited the Ca2+ signal. We conclude that uPAR associates with the CBD region of L-selectin to form a functional signaling complex.

Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcal Infections (PANDAS): Experience at a Tertiary Referral Center.

Background Pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS) is an autoimmune disorder presenting with obsessive compulsive disorder and/or tics. Like Sydenham’s chorea, its presumed pathogenesis consists of autoantibodies cross-reacting with neurons in response to a group A beta-hemolytic streptococcal infection (GASI). There are currently no diagnostic laboratory findings and management ranges from antibiotic prophylaxis to intravenous immunoglobulin to plasmapheresis. The diagnosis remains controversial, resulting in inconsistent referrals and significant patient anxiety. Methods A retrospective study was performed on all patients referred to the Pediatric Infectious Disease Division with a pre-referral diagnosis of PANDAS. Patients were analyzed by demographics, medical history, co-morbidities, symptoms, prior treatment, laboratory tests, management strategies, and treatment outcomes. Results From 2003 to 2013, there were 21 patients with a pre-referral diagnosis of PANDAS. Only five met the diagnostic criteria. No patient at referral had an objective scale to monitor symptoms. Eight referrals had a major psychiatric disorder, and none fulfilled diagnostic criteria (p<0.01). Discussion The majority of the patients referred with a pre-diagnosis of PANDAS do not fulfill diagnostic criteria nor do they have objective criteria for symptom monitoring. Major psychiatric disorders do not seem to be associated with PANDAS, and better physician education may prevent misdiagnoses. Multidisciplinary management is recommended.

A pediatric case of Cardiobacterium hominis endocarditis

Gram negative endocarditis is relatively rare in pediatrics but when they occur they are most frequently caused by one of the HACEK (Haemophilus species, Actinobacillus actinomycetemcomitans, C. hominis, Eikenella corrodens and Kingella kingae) group of microorganisms. Within the HACEK group of microorganisms there have been approximately 100 cases of Cardiobacterium hominis endocarditis reported in the literature, but only 2 previous cases of endocarditis and one case of pericarditis have been reported in children. In this report, we present a case of a 12-year-old boy with a right ventricle to pulmonary artery conduit for Tetralogy of Fallot with pulmonary atresia who presented at an annual cardiology examination with a 3 week history of fatigue and was found to have a vegetation on routine echocardiogram. Subsequent blood cultures grew Cardiobacterium hominis and the patient was treated successfully with 6 weeks of appropriate antibiotic therapy. We present this case and a review of the literature of the HACEK group of microorganisms in pediatrics.

Lemierre syndrome presenting as acute mastoiditis in a 2-year-old girl with congenital dwarfism

Lemierre syndrome is defined by septic thrombophlebitis of the internal jugular vein caused by Fusobacterium. Historically, these infections originate from the oropharynx and typically are seen in older children, adolescents and young adults. More recently, otogenic sources in younger children have been described with increasing frequency. We present a case of a two-year old, who initially developed an otitis media with perforation of the tympanic membrane and went on to develop mastoiditis and non-occlusive thrombosis of the venous sinus and right internal jugular vein. Fusobacterium necrophorum was grown from operative cultures of the mastoid, ensuing computed tomography scan revealed occlusion of the internal jugular vein and the patient was successfully treated with clindamycin, ciprofloxacin and enoxaparin. This case demonstrates the importance of considering Fusobacterium in otogenic infections and the consideration of Lemierre syndrome when F. necrophorum is identified

Antibiotic prescription patterns for management of acute otitis media in Lebanon

OBJECTIVES The high incidence of Acute Otitis Media (AOM) along with low antibiotic efficacy in the treatment of AOM is particularly favorable for the emergence of antimicrobial resistance. The promotion of more conservative antibiotic prescription habits has become an important focus of governments and academic societies. Little is known about the awareness and use of AOM practice guidelines by physicians in the Middle East. Our aim is to characterize AOM management in Lebanon by using an anonymous survey instrument to uncover potential disparities in treatment trends and evaluate differences in clinical guideline adherence patterns. METHODS A total of 75 practicing physicians were anonymously surveyed in Beirut, Saida, Nabatieh, Bekaa and Tripoli, Lebanon. The survey tool used was previously used in Amman, Jordan by our colleagues at the University of Michigan. The survey we used assessed awareness of and adherence to practice guidelines by prompting responses to hypothetical AOM cases. Differences in performance between various physician groups were noted. RESULTS Overall, physician participants answered 67% of the survey questions correctly. Trainees did better overall in terms of AOM management (62% correct responses as compared to 48% in attending physicians, p = 0.0175). Trainees also performed better in terms of their ability to manage cases of potential AOM in two-year old children and their ability to choose the appropriate medications (79% correct response rate compared to 71% in attending physicians, p = 0.0278). Participants who reported guideline adherence most or all of the time had a 67% correct response rate in regards to their ability to diagnosis AOM, compared to a 57% correct response rate in those who reported adhering sometimes or rarely to the guidelines (p = 0.0489). In the cases requiring antibiotic treatment for body temp of over 39C with/without otalgia, only 47-57% of participants identified the appropriate antibiotic regimen. CONCLUSION There are areas of potential improvement in adherence to clinical guidelines in the management, diagnosis, and treatment of AOM by Lebanese physicians. Conducting interventions among physicians to improve awareness of clinical guidelines and current treatment recommendations in Lebanon will likely improve adherence to guidelines, enhance clinical outcomes, and may help advance the fight against antimicrobial resistance.

School-based health education in Yucatan, Mexico about the Chikungunya virus and mosquito illness prevention

The Chikungunya virus (CHIKV) has been rapidly spreading throughout Latin America, utilizing pre-existing vectors to infiltrate the immunologically naïve populations. With the current rise of the Zika Virus, there is an urgent need for more rigorous vector control efforts to prevent further Zika breakout. We designed a school-based education module on CHIKV and mosquito prevention and presented it to the local students of ages of 6-18 in a rural town called Sudzal in Yucatan, Mexico. We distributed questionnaires before and after education to test the students’ knowledge of CHIKV and mosquito prevention. Chi-squared test was performed to determine the efficacy of the presentation in increasing their knowledge. The education presentation has proven to effectively educate the local residents in several critical methods of mosquito prevention, increasing the average test scores by 67% post-education. These include applying repellent, staying hydrated during recuperation, and cleaning indoor water containers to eliminate breeding sites (P<0.001). Furthermore, the questionnaire captured the residents’ behavioral patterns regarding CHIKV and mosquito prevention and identified cultural, ecological, and socioeconomic factors hindering effective implementation of vector control.

Disseminated Mycobacterium avium complex in an adolescent with perinatally-acquired HIV infection

Mycobacterium avium complex (MAC) is the most frequent nontuberculous mycobacteria implicated in opportunistic infections that define acquired immunodeficiency syndrome. With highly active antiretroviral therapy, disseminated MAC (dMAC) has become a rare entity. This unique case of dMAC was diagnosed in an adolescent with newly diagnosed perinatally-acquired HIV infection whose initial CD4 cell count was severely depleted and viral load was extremely high. While maximized treatment regimen had not been able to control his dMAC, improvement was noted when granulocyte macrophage colony-stimulating factor (GM-CSF) was added. GM-CSF should be considered as an adjunctive therapy in patients with refractory dMAC.

Management of Acute Osteomyelitis: A Ten-Year Experience

Osteomyelitis is an infection of the bone; proper management requires prolonged antibiotic treatment. Controversy exists as to when a patient should transition from intravenous to oral antibiotics. However, due to the high bioavailability of some oral antibiotics, optimal time to transition from high to low bioavailability antibiotics is a more valid consideration. Additionally, there are questions surrounding the efficacy of certain antibiotics, specifically trimethoprim-sulfamethoxazole (TMP-SMX), in treating osteomyelitis. After obtaining Institutional Review Board approval from both universities, a retrospective chart review was conducted, utilizing an author-created severity scale, on all patients seen by Pediatric Infectious Diseases at the Universities of Michigan and Toledo with an acute osteomyelitis diagnosis from 2002-2012. There were 133 patients, 106 treated successfully. Success was defined in this study specifically as treatment of <14 weeks without recurrence within 30 days of stopping antibiotics or permanent site disability. Seventeen patients were treated with TMP-SMX at comparable cure rates. Patients with pre-existing bone defects (noted in radiological reports), initial erythrocyte sedimentation rate (ESR)≥70, hematogenous osteomyelitis with soft tissue extension, and skull osteomyelitis were associated with increased failure rate. Switch to low bioavailability antibiotics occurred, on average, at 3.5 weeks; however, switching before then was not associated with decreased cure rate. As prevalence of methicillin-resistant Staphylococcus aureus (MRSA), especially clindamycin-resistant MRSA, increases, TMP-SMX appears to be an acceptable antibiotic. There does not appear to be a minimum length of high bioavailability treatment required for cure. Prior bone defect, extensive infection, ESR≥70, or skull osteomyelitis may be indications for more aggressive management.

Analysis of the Nature of IRB Contingencies Required for Informed Consent Document Approval

The University of Michigan Human Research Protection Program formed a six-member committee to analyze the nature of Institutional Review Board (IRB) staff and board contingencies for the approval of informed consent documents. Of the 100 studies examined, 87% had one or more informed consent contingencies. “Omissions” in documentation (40%) and “better clarity” (24%) accounted for the majority, while “word-smithing” accounted for only 10%. This is one of the first studies to examine the nature of IRB contingencies as they relate to informed consent documents. Educational efforts targeting completeness in documentation and clarity on the part of study teams, and discouraging “word-smithing” on the part of IRBs, could reduce the number of informed consent contingencies and expedite the IRB approval process.