Fibrinogen-neutrophil interactions in response to fMLP and Porphyromonas gingivalis fimbrial peptides

Porphyromonas gingivalis (P.g) is the primary bacterial agent in many forms of chronic periodontitis. Since polymorphonuclear leukocytes (PMNs) are first-line responders to P.g.- induced inflammation, and fibrinogen is important for in vivo PMN in this disease, we have studied the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP) (an inflammatory stimulus), P.g. fimbriae and fimbrial peptides (based on FimA, the main structural protein of P.g. fimbriae) on PMN-fibrinogen interactions. Freshly isolated human PMNs were allowed to react with FITC-Fibrinogen and various fimbrial peptides (denoted as FimA followed by amino acid number within whole FimA protein), and FITC-Fibrinogen binding was measured using flow cytometry. Freshly isolated neutrophils were also challenged with Fibrinogen and/or fimbrial peptides to measure IL-8 secretion using ELISA. Our studies show that fibrinogen binding to PMNs is enhanced (p < 0.01) in response to fMLP as well as fimbrial peptides (FimA 61-80) containing the motif LTTE (p < 0.01) in a dose dependent manner but not in response to peptides without that motif. We also observed that fMLP and FimA 61-80 have an additive effect on fibrinogen binding to PMNs (p < 0.05), and fMLP and FimA 171-185 significantly inhibit fMLP-induced fibrinogen binding (p < 0.01). To determine of the role of inflammatory cytokines, we examined IL-8 release from PMNs in response to combinations of P. gingivalis fimbriae, fMLP and fibrinogen. In all cases, IL-8 release increased in a dose-dependent manner (p < 0.05). fMLP-fibrinogen effect on IL-8 release from PMNs was synergistic while fimbriae-fibrinogen effect was additive. In summary, PMN priming by fimbrial peptides facilitates fibrinogen-PMN interaction and may increase inflammation.     

Traditional acupuncture in migraine: a controlled, randomized study

OBJECTIVE: To check the effectiveness of a true acupuncture treatment according to traditional Chinese medicine (TCM) in migraine without aura, comparing it to a standard mock acupuncture protocol, an accurate mock acupuncture healing ritual, and untreated controls. BACKGROUND: Migraine prevalence is high and affects a relevant rate of adults in the productive phase of their life. Acupuncture has been increasingly advocated and used in Western countries for migraine treatment, but the evidence of its effectiveness still remains weak. A large variability of treatments is present in published studies and no acupoint selection according to TCM has been investigated so far; therefore, the low level of evidence of acupuncture effectiveness might partly depend on inappropriate treatment. DESIGN AND METHODS: A prospective, randomized, controlled study was performed in 160 patients suffering from migraine without aura, assessed according to the ICD-10 classification. The patients were divided into the following 4 groups: (1) group TA, treated with true acupuncture (according to TCM) plus Rizatriptan; (2) group RMA, treated with ritualized mock acupuncture plus Rizatriptan; (3) group SMA, treated with standard mock acupuncture plus Rizatriptan; (4) group R, without prophylactic treatment with relief therapy only (Rizatriptan). The MIDAS Questionnaire was administered before treatment (T0), at 3 (T1) and 6 months (T2) from the beginning of treatment, and the MIDAS Index (MI) was calculated. Rizatriptan intake was also checked in all groups of patients at T0, T1, and T2. Group TA and RMA were evaluated according to TCM as well; then, the former was submitted to true acupuncture and the latter to mock acupuncture treatment resembling the same as TA. The statistical analysis was conducted with factorial ANOVA and multiple tests with a Bonferroni adjustment. RESULTS: A total of 127 patients completed the study (33 dropouts): 32 belonged to group TA, 30 to group RMA, 31 to group SMA, and 34 to group R. Before treatment the MI (T(0)) was moderate to severe with no significant intergroup differences. All groups underwent a decrease of MI at T(1) and T(2), with a significant group difference at both T(1) and T(2) compared to T(0) (P < .0001). Only TA provided a significant improvement at both T(1) and T(2) compared to R (P < .0001). RMA underwent a transient improvement of MI at T(1). The Rizatriptan intake paralleled the MI in all groups. CONCLUSIONS: TA was the only treatment able to provide a steady outcome improvement in comparison to the use of only Rizatriptan, while RMA showed a transient placebo effect at T1.     

Ex vivo-activated human macrophages kill chronic lymphocytic leukemia cells in the presence of rituximab: mechanism of antibody-dependent cellular cytotoxicity and impact of human serum

Antibody-dependent cellular cytotoxicity (ADCC) is one of the mechanisms of tumor killing during antibody (Ab) immunotherapy, and a role for myeloid cells as effectors has been observed in several models. We are developing immunotherapy approaches based on administration of large numbers of ex vivo interferon-gamma-activated macrophages to cancer patients. With a quantitative assay measuring killing of nonproliferating tumor cells, we evaluated whether, in physiologic conditions, these macrophages synergize with the anti-CD20 Ab rituximab for killing primary B-cell chronic lymphocytic leukemia (B-CLL) cells. ADCC reached levels of 70% to 80% at effector to target ratios as low as 1:1. Macrophage recruitment by Ab-opsonized tumor cells did not result in enhanced cytokine secretion, suggesting that the cytokine shower observed in rituximab-treated patients is not caused by macrophage activation, and that cytokines have no role in CLL killing. We observed that uptake of tumor material by macrophages was not directly correlated to tumor killing. Nonetheless, experiments in the presence of cytochalasin D showed that ADCC occurred mainly by phagocytosis. Tumor killing was largely mediated by Fc gammaRI and inhibited by increasing concentration of serum. Importantly, complement deposition on B-CLL cells did not seem to enhance macrophage ADCC in this model, as complement-depleted and complement-repleted human plasmas exerted comparable inhibition.     

Dissociable Control of Impulsivity in Rats by Dopamine D2/3 Receptors in the Core and Shell Subregions of the Nucleus Accumbens

Previous research has identified the nucleus accumbens (NAcb) as an important brain region underlying inter-individual variation in impulsive behavior. Such variation has been linked to decreased dopamine (DA) D2/3 receptor availability in the ventral striatum of rats exhibiting spontaneously high levels of impulsivity on a 5-choice serial reaction time (5-CSRT) test of sustained visual attention. This study investigated the involvement of DA D2/3 receptors in the NAcb core (NAcbC) and the NAcb shell (NAcbS) in impulsivity. We investigated the effects of a DA D2/3 receptor antagonist (nafadotride) and a DA D2/3 partial agonist (aripiprazole) infused directly into either the NAcbC or NAcbS of rats selected for high (HI) and low (LI) impulsivity on the 5-CSRT task. Nafadotride increased significantly the level of impulsivity when infused into the NAcbS, but decreased impulsivity when infused into the NAcbC of HI rats. By contrast, intra-NAcb microinfusions of aripiprazole did not affect impulsivity. Systemic administration of nafadotride had no effect on impulsive behavior but increased the number of omissions and correct response latencies, whereas systemic injections of aripiprazole decreased impulsive and perseverative behavior, and increased the number of omissions and correct response latencies. These findings indicate an opponent modulation of impulsive behavior by DA D2/3 receptors in the NAcbS and NAcbC. Such divergent roles may have relevance for the etiology and treatment of clinical disorders of behavioral control, including attention-deficit hyperactivity disorder and drug addiction.     

Risk factors for adverse outcomes of bacterial meningitis

Objective : To identify risk factors for adverse outcomes from bacterial meningitis.
Methodology : From a cohort of 166 children with bacterial meningitis who were studied prospectively, 130/158 (82%) survivors underwent neurological, neuropsychological, audiological and behaviour assessments 5–9 years following their illness.
Results : Major adverse outcomes included 8/166 (4.8%) deaths and severe neurological, intellectual or audiological sequelae in 11/130 (8.5%) children followed. Another 24 (18.5%) had cognitive, auditory or behaviour disorders. Bivariate analysis found age ≤12 months, tertiary referral, symptoms >24 h before diagnosis, seizures, focal neurological signs, deteriorating conscious state in hospital, Streptococcus pneumoniae infection and serum sodium concentration < 130 mmol/L were associated with adverse outcomes. Multivariate analysis showed age ≤12 months, symptoms >24 h, seizures after 72 h in hospital and focal neurological signs as independent risk factors. These were present in 18/19 (95%) children with major sequelae, but absent in 9/24 (37.5%) children with minor disabilities.
Conclusions : As minor disabilities following meningitis cannot be predicted, all survivors require assessment during their early school years.     

Electromyography and magnetic resonance imaging in the evaluation of radiculopathy

Electromyography (EMG) and magnetic resonance imaging (MRI) are commonly used in the diagnosis of cervical and lumbosacral radiculopathy, but the agreement between the two studies is unknown. We retrospectively studied 47 patients with a clinical history compatible with either cervical or lumbosacral radiculopathy who were evaluated with both an EMG and a spine MRI within 2 months of each other. Among these patients, 55% had an EMG abnormality and 57% had an MRI abnormality that correlated with the clinically estimated level of radiculopathy. The two studies agreed in a majority (60%) of patients, with both normal in 11 and both abnormal in 17; however, only one study was abnormal in a significant minority (40%), suggesting that the two studies remain complementary diagnostic modalities. The agreement was higher in patients with abnormal findings on neurologic examination, underscoring the difficulty of confirming the diagnosis in mild radiculopathy.     

Infusion of bispecific monoclonal antibody complexes into monkeys provides immunologic protection against later challenge with a model pathogen.

Heteropolymers (HP), bispecific mAbs which bind target pathogens to primate erythrocytes via complement receptor 1, facilitate clearance of pathogens from the bloodstream by targeting them for destruction in the liver without causing lysis or clearance of the erythrocytes. We show that when HP prepared with mouse IgG are intravenously infused into monkeys one or more times prior to exposure to a prototype pathogen, they bind to erythrocytes and remain in the circulation long enough to act as "sentinels," preventing pathogen invasion of the bloodstream. The effectiveness of HP as sentinels is limited both by the monkey's immune response to the HP and, prior to the immune response, by a gradual loss of the HP from monkey erythrocytes over a period of 1 week, and we have investigated possible causes of this HP loss. In overview, our results suggest that HP prepared with mouse IgG are able to effectively function as sentinels for a minimum of 4 days and, after repeat infusion, possibly for up to 2 weeks.     

Automation of a series of cutaneous topography measurements from silicon rubber replicas

Background/Aims Skin relief is a matter of interest for dermatologists and surgeons. One of the methods available for surface topography measurement is based on 3D profilometry using skin surface replicas. and most studies use statistical results obtained from a large number of skin replica samples. The advent of optical profilometers (without contact) made it possible to remove the solid positive replica and to reduce the duration of the profilometric data acquisition. Nevertheless this saving of time, to be really interesting, needs to automate the data acquisition on a series of negative replicas. Methods/Results By adding a video camera to the optical profilometer and then by processing the resulting images, we have conceived a system able to carry out topographic measurements on a series of replicas loosely organized on a sample holder, without any human intervention. The silicon replicas in use have a very light colour: nearly white, sometimes slightly blue or green. The laser spot of the profilometer is so luminous that its red colour looks white through the camera. When choosing a replica holder with a matt dark colour and marking the left upper corner of the study area on the replica in black ink, the colours to be differentiated on the image are then close to the black one and the white one. We accordingly change the colour camera image into a black and white image (with 256 grey levels) and then carry out thresholdings to separate the different objects or information included in this image. With the use of a perfectly circular replica, of an accurately known size, laid on the sample holder at the center of the area filmed by the camera, we adjust the threshold level, which allows separation of the replica from its holder. We then move this calibrated replica in order to find the relationship between the size in pixels and the real size on the sample holder, in various positions of the video image. The software has four main built-in stages:

• Moving the sample holder beneath the sensor until a part of a replica is detected in the field of view of the camera;
• Moving the sample holder until this replica lies just in the middle of the image given by the camera;
• Recognition of the mark of the upper left corner of the surface area to be measured out inside this replica; and
• Moving the sample holder until the laser spot of the profilometer coincides with the origin of the surface area to be measured out, then carrying out this measurement.

From the upper left corner of the sample holder, a scanning, line-by-line or column-by-column (according to the selected priority direction), is carried out until the successive replicas are found, and is stopped as soon as the number of replicas entered by the operator is reached. Conclusion The simplicity of the algorithms used makes it possible to distinguish the next measurement area from the preceding one in a few seconds.