Intracellular parasitism by the human granulocytic ehrlichiosis bacterium through the P-selectin ligand, PSGL-1.

Human granulocytic ehrlichiosis (HGE) is a febrile tick-borne illness caused by a recently discovered intracellular bacterium remarkable for its tropism for professionally phagocytic neutrophils. Monoclonal antibodies against the P-selectin binding domain of the leukocyte P-selectin glycoprotein ligand, PSGL-1, prevented HGE cell binding and infection, as did enzymatic digestion of PSGL-1. Furthermore, simultaneous neoexpression in nonsusceptible cells of complementary DNAs for both PSGL-1 and its modifying alpha-(1,3) fucosyltransferase, Fuc-TVII, allowed binding and infection by HGE. Thus, the HGE bacterium specifically bound to fucosylated leukocyte PSGL-1. Selectin mimicry is likely central to the organism's unique ability to target and infect neutrophils.

Potentiated adherence of sickle erythrocytes to endothelium infected by virus.

Systemic viral infection is a known precipitant of vasocclusive crisis in sickle patients, but the mechanism underlying this clinical observation is unknown. In the present studies, human umbilical vein endothelial cells were infected with Herpes simplex virus type 1 (HSV) to model systemic viral disease. The already abnormal adherence of sickle erythrocytes to control endothelium is enhanced 1.8 +/- 0.4-fold to HSV-infected endothelium (P less than 0.001). This component of potentiated adherence is eliminated by maneuvers that block Fc receptors, it is prevented by tunicamycin, and it is not seen using a mutant HSV that is unable to express the Fc receptor glycoprotein. Thus, the incremental adherence seen here occurs due to expression of Fc receptor activity on HSV-infected endothelium and the consequent recognition of abnormal amounts of IgG on sickle erythrocytes. We conclude that systemic viral infection potentially can induce a novel mechanism for enhancement of erythrocyte adherence to endothelium and that this may increase the likelihood of vasocclusion during viral infection.

Leukocyte infection by the granulocytic ehrlichiosis agent is linked to expression of a selectin ligand.

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne illness caused by an intracellular bacterium that infects neutrophils. Cells susceptible to HGE express sialylated Lewis x (CD15s), a ligand for cell selectins. We demonstrate that adhesion of HGE to both HL60 cells and normal bone marrow cells directly correlates with their CD15s expression. HGE infection of HL60 cells, bone marrow progenitors, granulocytes, and monocytes was blocked by monoclonal antibodies against CD15s. However, these antibodies did not inhibit HGE binding, and anti-CD15s was capable of inhibiting the growth of HGE after its entry into the target cell. In contrast, neuraminidase treatment of HL60 cells prevented both HGE binding and infection. A cloned cell line (HL60-A2), derived from HL60 cells and resistant to HGE, was deficient in the expression of alpha-(1, 3)fucosyltransferase (Fuc-TVII), an enzyme known to be required for CD15s biosynthesis. Less than 1% of HL60-A2 cells expressed CD15s, and only these rare CD15s-expressing cells bound HGE and became infected. After transfection with Fuc-TVII, cells regained CD15s expression, as well as their ability to bind HGE and become infected. Thus, CD15s expression is highly correlated with susceptibility to HGE, and it, and/or a closely related sialylated and alpha-(1,3) fucosylated molecule, plays a key role in HGE infection, an observation that may help explain the organism's tropism for leukocytes.

Relationships between protein structure and dynamics from a database of NMR-derived backbone order parameters.

The amplitude of protein backbone NH group motions on a time-scale faster than molecular tumbling may be determined by analysis of (15)N NMR relaxation data according to the Lipari-Szabo model free formalism. An internet-accessible database has been compiled containing 1855 order parameters from 20 independent NMR relaxation studies on proteins whose three-dimensional structures are known. A series of statistical analyses has been performed to identify relationships between the structural features and backbone dynamics of these proteins. Comparison of average order parameters for different amino acid types indicates that amino acids with small side-chains tend to have greater backbone flexibility than those with large side-chains. In addition, the motions of a given NH group are also related to the sizes of the neighboring amino acids in the primary sequence. The secondary structural environment appears to influence backbone dynamics relatively weakly, with only subtle differences between the order parameter distributions of loop structures and regular hydrogen bonded secondary structure elements. However, NH groups near helix termini are more mobile on average than those in the central regions of helices. Tertiary structure influences are also relatively weak but in the expected direction, with more exposed residues being more flexible on average than residues that are relatively inaccessible to solvent.

Pneumocystis with normal chest X-ray film and arterial oxygen tension. Early diagnosis in a patient with the acquired immune deficiency syndrome.

A patient with Kaposi's sarcoma and the acquired immune deficiency syndrome became acutely febrile and dyspneic. Although chest roentgenograms and findings from arterial blood oxygenation studies were normal, bronchoscopy disclosed heavy Pneumocystis carinii infection. The patient was treated with trimethoprim-sulfamethoxazole with a rapid clinical response. It has been distinctly unusual to diagnose Pneumocystis without roentgenographic or blood gas abnormalities. Pneumocystis infection probably occurs as a wide spectrum of disease ranging from subclinical infection to frank pneumonitis. In the appropriate clinical setting, clinically significant Pneumocystis infection may be diagnosed despite the absence of an infiltrate or hypoxemia and early treatment may be beneficial.

Bloodstream invasion in early Lyme disease: results from a prospective, controlled, blinded study using the polymerase chain reaction.

PURPOSE: The purposes of this study were to determine (1) the optimal techniques for and potential diagnostic usefulness of the polymerase chain reaction (PCR) in early Lyme disease, and (2) the true frequency and clinical correlates of PCR-documented blood-borne infection in the dissemination of Lyme disease. PATIENTS AND METHODS: We performed a prospective, controlled, blinded study of PCR, culture, and serology on fractionated blood samples from 105 patients; 76 with physician-diagnosed erythema migrans and 29 controls. Clinical characteristics of the patients were obtained with a standardized data entry form and correlated with results of the laboratory studies. RESULTS: Only 4 of the 76 (5.3%) patients with erythema migrans were culture positive; however, 14 of 76 (18.4%) had spirochetemia documented by PCR of their plasma. None of 29 controls were PCR or culture positive (P = 0.007, versus patients). PCR-documented spirochetemia correlated with clinical evidence of disseminated disease; 10 of 33 patients (30.3%) with systemic symptom(s) were PCR positive compared to 4 of 43 (9.3%) without such evidence (P = 0.02). PCR positivity was more frequent among patients with each of four specific symptoms: fever, arthralgia, myalgia, and headache (all P < 0.05). A higher total number of symptoms (median 2.5 in PCR-positive patients versus 0 in PCR-negative controls; P < 0.01) and the presence of multiple skin lesions (37.5% of patients with multiple, versus 13.3% of patients with single lesions [P = 0.04] were also correlated with PCR positivity. Patients with both systemic symptoms and multiple skin lesions had a 40% PCR-positivity rate; however, 4 of 42 (9.5%) asympatomatic patients with only single erythema migrans lesions were also PCR positive. In multivariate analysis using logistic regression, the number of systemic symptoms was the strongest independent predictor of PCR positivity (P = 0.004). CONCLUSIONS: PCR detection of Borrelia burgdorferi is at least three times more sensitive than culture for identifying spirochetemia in early Lyme disease and may be useful in rapid diagnosis. PCR positivity significantly correlates with clinical evidence of disease dissemination. Bloodstream invasion is an important and common mechanism for the dissemination of the Lyme disease spirochete.

Possible transmission of herpes simplex virus by organ transplantation.

Herpes simplex virus commonly reactivates in seropositive transplant recipients but has not been generally thought to be transmissible by the transplanted organ itself. We studied two consecutive cases of disseminated HSV, without mucosal lesions, occurring in a heart and in a pancreatic transplant recipient, and implicate the allografts as the source of the virus. In both cases the recipients were seronegative pretransplant by complement fixation (less than 1:4), neutralization (less than 1:2), and complement enhanced neutralization (less than 1:4), and by radioimmunoprecipitation of HSV-2 antigens with serum followed by polyacrylamide gel electrophoresis (RIPA-PAGE). Both recipients' isolates were HSV-2 by restriction mapping and each donor had antibodies directed specifically against HSV-2, as determined by differential neutralization (HSV-2/HSV-1 ratios 1.46 and 1.58, where greater than 0.85 indicates antibody to HSV-2). Posttransplant, each recipient developed an antibody response with temporal antigenic specificity and complement-enhanced neutralization consistent with primary infection. These findings have important clinical and pathogenic implications and suggest that latent or reactivated HSV-2 DNA transplanted in donor tissues may cause severe infection in seronegative and immunosuppressed transplant recipients.

Passage of herpes simplex virus type 1 on chick embryo fibroblasts confers virulence for chick embryos.

The pathogenesis of chorioallantoic membrane (CAM) infection with herpes simplex virus 1 and 2 (HSV-1 and HSV-2) as well as chick embryo fibroblast (CEF) passaged HSV-1 was studied. It was found that HSV-2 is at least a million-fold more virulent than HSV-1 as measured by pfu/LD50 ratios for the embryo. Serial passage of HSV-1 in vitro on CEF cells selected for a virus (CEFP10) which, unlike its parental HSV-1 strain, is able to kill the embryo. The restriction endonuclease maps of CEFP10 and its parental strain are indistinguishable and the mechanism of the increased virulence of CEFP10 was demonstrated to be its enhanced replication in chick embryo cells both in vivo and in vitro. In contrast, despite its inferior replicative ability, HSV-2 was found to have a biologically important specific invasiveness function that is not simply related to overall viral replication. Finally, the ability to isolate HSV-1 (CEFP10) virulent for the chick embryo after passage in vitro illustrates that tissue culture passage of HSV in appropriate cells may actually increase virulence for the animal host.

Induction of tissue factor procoagulant activity in myelomonocytic cells inoculated by the agent of human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is a recently recognized rickettsial tick-borne febrile illness that may occasionally be complicated by coagulopathy. The agent of HGE (aHGE) is an obligate intracellular pathogen, which replicates in endosomes within neutrophils and their precursors. We hypothesized that aHGE might cause DIC via induction of monocyte tissue factor procoagulant activity (TF PCA). Peripheral blood mononuclear cells (PBMNC) and HL-60 cells were used to model the effect of aHGE infection on monocytes/macrophages. Mononuclear cells inoculated with aHGE in vitro demonstrated approximately a 12-15-fold increase in TF PCA, with peak activity occurring at 8-12 h. HL-60 cells inoculated with aHGE also manifested a 4-6 fold induction of TF PCA, with maximal activity occurring at about 8 h. By comparison, E. Coli lipopolysaccharide (LPS) also induced an increase in TF PCA of an equivalent magnitude, and with a similar time course. Induction of TF did not require inoculation of HL-60 cells with live organism, since heat-inactivated aHGE still stimulated TF PCA expression in the target cells. Furthermore, filtered supernatants from heat-inactivated organisms induced TF PCA suggesting that the effect is due to a soluble mediator produced by the organism. Although aHGE is a gram negative organism, the soluble mediator did not appear to be classic endotoxin in that the supernatants tested negative for endotoxin by the Limulus Amoebocyte assay, and polymixin had no inhibitory effect on aHGE supernatants. We conclude that aHGE induces cells of the myelo-monocytic lineage to synthesize TF, which may contribute to the clinical coagulopathy that can be observed in this condition. An atypical soluble mediator or cellular component of the organism appears to be critically important in TF induction by aHGE.

A prospective study of the seroprevalence of Borrelia burgdorferi infection in patients with severe heart failure.

We conclude that Lyme disease is not a common cause of idiopathic heart failure in the Midwestern United States and that false-positive Lyme disease serologic results are not rare among patients with severe heart failure. Patients with significant cardiac disease who are found to be EIA seropositive should have confirmatory Western blots performed before consideration of treatment. Based on our findings, we cannot recommend either the routine serologic screening of patients with idiopathic cardiomyopathy or aggressive (e.g., parenteral) antibiotic treatment of seropositive patients unless the specific clinical history suggests antecedent Lyme disease.

Characterization of monoclonal antibodies to an immunodominant protein of the etiologic agent of human granulocytic ehrlichiosis.

Immunodominant proteins in the range of 42-45 kD are important for the serodiagnosis of human granulocytic ehrlichiosis (HGE). Antigens from human isolates of the etiologic agent of HGE cultivated in HL-60 cells were used to immunize BALB/c mice and generate a panel of hybridomas secreting monoclonal antibodies. Using an enzyme immunoassay, an immunofluorescent assay (IFA), and Western blotting, we showed that culture supernatants and ascites of these hybridomas were reactive with human isolates of the etiologic agent of HGE, Ehrlichia equi and E. phagocytophila. Following screening and subcloning, we selected three stable hybridomas, R1B10, R5E4, and R5A9, which were determined to be of the isotypes IgG3, IgG1, and IgG2a, respectively. These results suggest that the epitopes of the 42-45-kD protein recognized by these three monoclonal antibodies are conserved among E. equi, E. phagocytophila, and the etiologic agent of HGE. Western blot analysis showed reactivity with the 44-kD protein of human isolates of the HGE agent. None of the monoclonal antibodies were reactive with HL-60 cells that were not infected with the HGE agent. No cross-reactivity with related intracellular pathogens could be detected when undiluted supernatants from hybridoma cultures were allowed to react by IFA with antigens from E. chaffeensis, E. risticii, E. platys, Rickettsia rickettsii, R. prowazekii, or Coxiella burnetii. The additivity index of two antibodies, R5E4 and R1B10 was near zero, suggesting that these two antibodies may compete for the same epitope of the 44-kD protein, while monoclonal antibody R5A9 appears to interact with a different epitope. The antibodies secreted by these hybridomas may be useful as immunologic agents in serodiagnostic, immunohistochemical, and other studies of the etiologic agent of HGE.

Ocular involvement in an outbreak of herpes gladiatorum.

An epidemic of herpes simplex virus type 1 occurred in 60 of 175 wrestlers (34%) attending a four-week intensive training camp. Five of these 60 patients (8%) developed ocular involvement that included follicular conjunctivitis, blepharitis, and phlyctenular disease. Cultures of the conjunctiva and eyelid vesicles were positive for herpes simplex virus type 1 in four of the five patients with ocular disease. The viral isolates were compared by restriction-endonuclease analysis, which disclosed that three of the four isolates were the same strain. None of the patients had corneal involvement and there has been no evidence of viral recurrence to date. Herpes simplex virus type 1 is a health risk for wrestlers, and ocular infections are part of the clinical spectrum. Prompt diagnosis and appropriate management of the outbreak may reduce the severity of the outbreak transmission.

Energy expenditure of young Swazi women as measured by the doubly-labelled water method.

The energy expenditure of a group of young (age 18-28 years) Swazi women resident at an agricultural college was measured using the doubly-labelled water (DLW) method. This was a baseline study to evaluate feasibility of the protocol under field conditions in Swaziland and to determine energy needs of sedentary women under well controlled conditions. Measurements were based on saliva samples collected over a 10-d period using the two-pair method. Two pairs of data points (days 2-9 and 3-10) were used to calculate expenditure. The two sets of data points were within 2.5 per cent of each other. Dietary intake was measured by self-report diaries for 5 study days and was not significantly different from the DLW measurements. Energy expenditure averaged 1735 kcal (7252 kJ)/d and 44 kcal (185 kJ)/kg fat-free mass, lower than other groups similarly measured. Intake averaged 1738 kcal (7265 kJ)/d. Low levels of expenditure observed probably reflect inactivity.

Herpes virus infection of endothelium: new insights into atherosclerosis.

Several pieces of evidence suggest that vascular endothelium may be a site of latent herpetic viral infection, and that activation of such infection might cause or aggravate atherosclerosis. The present studies which utilized HSV-1 infection of cultured endothelial monolayers, provide insights into two phenomena seemingly relevant in considerations of atherosclerosis. Thus, mechanisms are reported by which infected endothelium may be damaged by marginated inflammatory cells, and be transformed from an anticoagulant to a procoagulant tissue. First, granulocytes are attracted to, and avidly bind, endothelium infected for very brief periods. This interaction is associated with denudation of intact cells as well as actual cytolysis through release of PMN proteases and toxic oxygen species. Second, several potentially additive abnormalities of HSV-infected endothelium would seem to foster coagulation. These include: a) its loss of surface heparans and thrombomodulin; b) its inability to synthesize prostacyclin with associated incapacity to deter platelet adhesion; c) its disordered membrane lipid conformation which is likely associated with excessive surface thrombin generation; and d) its unique ability to generate and release tissue factor. We speculate that mechanical abrasion may reactivate latent herpes (HSV or CMV) infection in endothelial cells particularly those exposed to high shear forces--for instance, at vessel bifurcations. This may underlie the endothelial damage, clotting and atheroma formation commonly found at these sites.

Epidemiological aspects of human granulocytic Ehrlichiosis in southern Germany.

Human granulocytic Ehrlichiosis (HGE) is a newly emerging acute febrile illness which is likely transmitted by ticks of the Ixodes ricinus/I. persulcatus complex. First seroepidemiological surveys on the prevalence of HGE antibodies, detection of DNA of granulocytotropic Ehrlichiae in I. ricinus and one case of HGE from Slovenia confirmed by serology and PCR (polymerase chain reaction) suggest that HGE might exist all over Europe. The purpose of the present study was a) to determine the prevalence of antibodies against the HGE agent in sera collected from persons at high risk for exposure to I. ricinus with that of a control population and b) to determine the prevalence of granulocytic Ehrlichiae in I. ricinus ticks from Southern Germany. We studied sera from 150 forestry workers and 105 patients with an established diagnosis of Lyme disease as tick-exposed populations. Sera from 103 healthy blood donors without a history of known tick bites served as controls. A significantly higher prevalence of HGE antibodies (P < or = 0.01) was present among patients with Lyme borreliosis (12 of 105 were positive; 11.4%) and forestry workers (21 of 150 were positive; 14%) compared to blood donors (2 of 103 were positive; 1.9%). Furthermore, 510 adult and nymphal I. ricinus were investigated by PCR for the presence of granulocytic Ehrlichiae with primers specific for the E. phagocytophila group. In eight (1.6%) of the investigated ticks the expected amplification product was detectable, indicating a low prevalence of infected ticks especially when compared with B. burgdorferi. The presented data strongly suggests that the HGE agent or a closely related organism exists in Southern Germany and therefore HGE should be considered in the differential diagnosis of febrile illnesses. However, final evidence can be provided only after isolation of the organism from patients.

Joint pain and swelling: could it be lyme arthritis?

This report presents two probable cases of Lyme arthritis triggered by athletic activity or trauma. One patient had ankle swelling after he began a jogging program, and a second had knee swelling that followed a twisting injury. The report describes the three stages of the illness and presents diagnostic lab tests and the therapies used. Polymerase chain reaction (PCR) testing of the synovial fluid can be useful in diagnosing active Lyme arthritis, but it may yield a false-negative result when there has been recent remission of the arthritis.

Transforming regulatory science 2012: making a difference.

"What does not change is the will to change."-Charles Olson, "The Kingfishers"The world is in the midst of scientific revolutions that can transform medicine and public health. Yet translation to needed products remains slow and expensive. There are major opportunities for new regulatory science to help transform product development and evaluation. A plan by the US Food and Drug Administration (FDA), "Advancing Regulatory Science," identifies eight priorities and numerous actions to help catalyze transformation. Scientific excellence and collaboration, including public and private sectors, are essential for change that benefits health and economies globally.