Antimicrobial Activity of a Cationic Guanidine Compound against Two Pathogenic Oral Bacteria.

This study evaluated the potential antimicrobial properties of a polyguanidine (CatDex) on two oral bacteria. Chlorhexidine gluconate 1340 µmoL L-1 (CHX 0.12%) was used as control. Streptococcus mutans (S. mutans) and Porphyromonas gingivalis (P. gingivalis) were grown in BHI media. Bacterial sensitivity and antimicrobial activity were determined by the minimum inhibitory concentration (MIC) and Kirby-Bauer methods. To study side effects, that is, toxicity, dental pulp stem cells (DPSCs) were used. Fluorometric cytotoxicity and confocal microscopy assays were used in order to test cell viability. CatDex inhibited growth of S. mutans at all concentrations and growth of P. gingivalis at all concentrations except 25 µmoL L-1. The MIC of CatDex was 50 µmoL L-1 for both S. mutans and P. gingivalis. The inhibition of bacteria exposed for 8 h at 50 µmoL L-1 of CatDex exhibited increased antimicrobial activity over time, with 91% inhibition in both bacteria. The antimicrobial activities of CatDex and CHX were similar when tested on two common bacteria. CatDex was significantly less toxic to DPSCs. CatDex toxicity depended on time and not on concentration. With regard to clinical relevance, CatDex may have potential as a novel antimicrobial agent. Further studies are in progress.

Immunomodulatory effect of diethylcarbamazine in mice infected with Nocardia brasiliensis.

We tested whether diethylcarbamazine (DEC) or ivermectin (IVM), both antiparasitic drugs with reported immunomodulatory properties, were able to affect the immune system to potentiate host defense mechanisms and protect against actinomycetoma in a mouse model. Male BALB/c mice of 10-12 weeks of age were injected with either Nocardia brasiliensis or saline solution. Recorded were the effects of a treatment by DEC (6 mg/kg per os daily for one week) or IVM (200 µg/kg subcutaneously on days 1 and 3) on (i) the development of mycetoma lesion, (ii) the expression of reactive oxygen intermediates (ROI) by phagocytes, (iii) the proliferation index of lymphocytes and (iv) antibody production of IgG and IgM. After an initial lesion in all mice, DEC inhibited a full development and progression of actinomycetoma resulting in a reduced lesion size (p < 0.001). IVM had no inhibitory effect on the development of mycetoma. Furthermore, DEC treatment was associated with a significant enhancement of ROI expression (p < 0.05) by polymorphonuclear neutrophils at day 3 after infection. Lymphocyte proliferation in response to N. brasiliensis antigens and concanavalin A in DEC-treated group was higher than in non-treated group at day 21 and 28 postinfection (p < 0.01). Significant changes in antibody response were not observed. By all parameters tested, DEC was superior to IVM regarding immunostimulatory potency. In conclusion, DEC expressed an in vivo influence on the immune status during the infection by N. brasiliensis leading to retrogression of the mycetoma and increasing cellular immune responses. Our findings may indicate a potential use of DEC as a putative adjuvant in infectious disease or vaccination.

A relationship between acute respiratory illnesses and weather.

Weekly data from 7 years (2004-2010) of primary-care counts of acute respiratory illnesses (ARIs) and local weather readings were used to adjust a multivariate time-series vector error correction model with covariates (VECMX). Weather variables were included through a partial least squares index that consisted of weekly minimum temperature (coefficient = - 0·26), weekly median of relative humidity (coefficient = 0·22) and weekly accumulated rainfall (coefficient = 0·5). The VECMX long-term test reported significance for trend (0·01, P = 0·00) and weather index (1·69, P = 0·00). Short-term relationship was influenced by seasonality. The model accounted for 76% of the variability in the series (adj. R 2 = 0·76), and the co-integration diagnostics confirmed its appropriateness. The procedure is easily reproducible by researchers in all climates, can be used to identify relevant weather fluctuations affecting the incidence of ARIs, and could help clarify the influence of contact rates on the spread of these diseases.

Effects of pentoxifylline on the urinary protein excretion profile of type 2 diabetic patients with microproteinuria: a double-blind, placebo-controlled randomized trial.

AIMS: The purpose of this study was to identify the effect of pentoxifylline on the urinary protein excretion profile in type 2 diabetic patients. METHODS: 40 type 2 nonhypertensive diabetic patients were randomly allocated to receive either pentoxifylline 400 mg t.i.d. or placebo daily for 16 weeks. Eligible subjects were those with urinary albumin excretion between 20 and 200 microg/min. Subjects receiving angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), calcium antagonists, and diuretics as well as those with reduced renal function, pregnancy, urinary tract infection, and smoking were not included. A 6-month pretreatment stabilization phase aimed to reduce and stabilize fasting serum glucose levels was carried out. Urinary proteins were identified by electrophoresis, and immunodetection was identified by Western blot. Electrophoretic analysis was performed using molecular weight markers of 150, 132, 77, and 66 kDa to identify high-weight proteins, and 54, 41, 36, 27, 21, 14.3, and 12 kDa to identify low-weight proteins. RESULTS: At baseline, subjects in both groups who showed a glomerular tubular pattern did not differ in their urinary excretion profile. The urinary proteins identified were immunoglobulin G, ceruloplasmin, transferrin, and albumin (glomerular pattern) as well as alpha1-antitrypsin, alpha1-acid glycoprotein, collagenase inhibitor, alpha1-microglobulin, trypsin inhibitor, lysozyme, and beta2-microglobulin (tubular pattern). Subjects who received pentoxifylline had reduced urinary excretion of high- and low-molecular weight proteins. CONCLUSIONS: Urinary protein excretion in type 2 diabetic subjects shows a mixed, glomerular and tubular, pattern. Pentoxifylline reduces the excretion of both high and low molecular-weight urinary proteins.

Differences in eosinophil and neutrophil chemotactic responses in sowda and generalized form of onchocerciasis.

Extravasation of host's leukocytes from blood vessels into inflammatory tissues represents a prerequisite for a subsequent interaction with invaded parasites. The migratory responses of eosinophilic and neutrophilic granulocytes in the polar forms of the filarial infection onchocerciasis were investigated. The hyporeactive, generalized form, the chronic hyperreactive (sowda) form and persons without signs of onchocerciasis from a hypoendemic area for onchocerciasis were compared. Eosinophils from sowda patients responded more strongly to the inflammatory mediator platelet-activating factor (PAF) than those from generalized patients and persons without onchocerciasis. The most significant differences were found between the sowda group and a subgroup of the generalized form with 16-80 microfilariae/mg skin (P<0.05) while patients with low microfilarial density exhibited chemotactic responses similar to the sowda group. In contrast to the strong eosinophil response, neutrophils from sowda patients appeared unreactive to PAF and the tripeptide activator formyl-methionyl-leucyl-phenylalanine (fMLP), thereby differing from both other groups. Laboratory data confirmed a state of hyperreactivity in sowda patients similarly found in generalized patients with low microfilarial density and contrasting with those with a microfilarial density of 16-80 mf/mg. The results revealed an inverse chemotactic responsiveness of eosinophil and neutrophil granulocytes in sowda patients and strengthened the observation of a spectrum of host's responsiveness in onchocerciasis.

Improved Randolph stain for direct leukocyte differentiation and determination of total eosinophil count in a hemocytometer.

Identification and quantification of eosinophilic granulocytes are commonly performed indirectly by total leukocyte count and white cell differentiation in smears or cytocentrifuge preparations. Using a combination of four dyes, phloxine, Biebrich scarlet, methylene blue, and crystal violet, at 50-800 micrograms/ml, we have substantially improved an earlier method for differentiating leukocytes in a hemocytometer. This direct method allowed a rapid and reliable enumeration of eosinophils and their differentiation from neutrophils, lymphocytes and monocytes in peripheral blood and leukocyte fractions. The results obtained using this stain correlated with the leukocyte counts calculated from May-Grünwald-Giemsa stained smears in 100 patients with eosinophilia of various etiologies (r = 0.95; p < 0.01). This simple method is a useful improvement for eosinophil enumeration in field studies and biological experiments where the purity of cell suspensions must be evaluated without delay. The method cannot be substituted for the commonly used indirect technique which also allows the identification of other leukocyte lineages and their precursors.

Modulatory effects of antifilarial drugs ivermectin, CGP 6140 and CGP 20376 on the oxidative burst of eosinophilic granulocytes.

Three recently introduced anthelmintic agents, the macrocyclic lactone, ivermectin, the amoscanate derivative CGP 6140 and the benzothiazole compound CGP 20376, were investigated for their in vitro modulatory effects on eosinophilic effector cells. The investigation comprised studies on the generation of the toxic oxygen intermediates superoxide anion and hydrogen peroxide which are major effector products of granulocytes. Eosinophils were obtained from 19 patients infected with the filarial parasite Onchocerca volvulus. Inhibitory effects on the generation of toxic oxygen intermediates were demonstrated for ivermectin and CGP 20376 at concentrations higher than 200 ng/ml (0.5 microM) and for CGP 6140 at concentrations higher than 1000 ng/ml (2.7 microM). An increased production of the reactive oxygen metabolites was demonstrated at low doses of ivermectin (20-40 ng/ml; corresponding to 0.02-0.04 microM) and CGP 6140 (40-100 ng/ml; 0.1-0.3 microM), respectively. The results reveal a dual, dose-dependent modulatory in vitro effect of the investigated anthelmintic drugs on the respiratory burst of eosinophilic effector cells indicating that these compounds may modulate host defense in vivo.

Eosinophil-larval-interaction in onchocerciasis: heterogeneity of in vitro adherence of eosinophils to infective third and fourth stage larvae and microfilariae of Onchocerca volvulus.

Adherence of eosinophilic granulocytes from patients with onchocerciasis to microfilariae (Mf), third (L3) and fourth (L4) stage larvae of Onchocerca volvulus was studied in vitro. Native and heat-inactivated sera from patients with onchocerciasis (OS), from endemic controls without signs of the disease (ECS), from healthy Caucasians (NS) or foetal calf serum (FCS) served as sources for adherence mediating factors. In FCS-supplemented medium eosinophils did not adhere to any larvae. None of the sera mediated the adherence of eosinophils to L4. Eosinophils adhered to L3 in the presence of OS, ECS and NS, whereas OS exclusively mediated adherence to Mf. Reduced adherence rates of eosinophils to L3 occurred in heat-inactivated or zymosan-activated OS, ECS or CS. Eosinophils bound to the L3 cuticle of moulting stage but not to the newly exposed L4 cuticle. A single adherent layer of effector cells was found around cast L3 cuticle, multiple layers were found around intact L3 leading to subsequent paralysis of the larvae and to an amplification of the toxic effector potential by homotypic intereosinophilic adhesion. Our experiments document heterogeneity of in vitro effector cell adherence to the three larval stages of O. volvulus and indicate that complement-dependent as well as independent mechanisms are operative in eosinophil-larval-interaction. The results emphasize the importance of the invading infective larval stages of O. volvulus as possible targets for vaccine production.

Serum-dependent interaction of granulocytes with Onchocerca volvulus microfilariae in generalized and chronic hyper-reactive onchocerciasis and its modulation by diethylcarbamazine.

The adherence and cytotoxicity of granulocytes to microfilariae of Onchocerca volvulus were examined in vitro. Reactivity and modulation by diethylcarbamazine of isolated eosinophilic and neutrophilic granulocytes from patients with generalized and chronic hyper-reactive onchocerciasis (sowda or localized form) from endemic foci in Liberia were evaluated under varying serum conditions. In the presence of pooled sera from patients with generalized onchocerciasis granulocytes from both polar groups of patients exhibited similar adherence rates, whereas immobilization rates were higher for eosinophils than for neutrophils. In localized onchocerciasis, the use of autologous serum resulted in a significant decrease in adherence and immobilization rates for both eosinophils and neutrophils. After preincubation of eosinophils, but not of microfilariae, with diethylcarbamazine autologous serum-mediated adherence and cytotoxicity were enhanced to rates similar to those found with pooled serum from individuals with generalized onchocerciasis. These results suggest that granulocytes from both forms of onchocerciasis did not differ with respect to their anti-parasitic reactivity and that antibodies as well as additional serum factors appear to contribute to the functional activity of these effector cells. The findings indicate that predominantly eosinophils, compared to neutrophils, damage the larvae of O. volvulus and support earlier observations which suggest that diethylcarbamazine influences the effector cells rather than the parasite itself.

Comparative study of eosinophil purification on Nycodenz, Metrizamide and Percoll density gradients.

Studies on the metabolism of human eosinophils and their interaction with helminthic parasites have been hampered by the difficulty in obtaining these granulocytes in sufficient purity and quantity. In this report, we describe for the first time the application of the density medium Nycodenz for the purification of eosinophils from peripheral blood. Nycodenz density gradient centrifugation is compared with Percoll and metrizamide gradient centrifugation with special emphasis on cell yield, purity of cell suspensions, and functional integrity of separated cells. Percoll gradients provide purer preparations (mean value 94 +/- 15%) of eosinophils from blood with moderate eosinophilia than Nycodenz or Metrizamide (58 +/- 29 and 88 +/- 13%, respectively). The recovery rates of eosinophil-enriched fractions were low after Nycodenz centrifugation (29 +/- 19%) in comparison to Metrizamide (60 +/- 21%) or Percoll (70 +/- 16%). Eosinophils (as neutrophils), prepared by the 3 techniques, function metabolically (superoxide anion generation) and in assays measuring phagocytosis (S. aureus, zymosan particles). Percoll gradient isolated eosinophils (as neutrophils) adhere to and immobilize larvae of Onchocerca volvulus.

Rapid method for the purification of viable microfilariae from nodules of Onchocerca volvulus by Percoll gradient centrifugation.

Microfilariae of O. volvulus were purified from dissected nodules by centrifugation on discontinuous Percoll density gradients. Nodules were used freshly or after cryopreservation in liquid nitrogen. Highly purified and motile microfilariae were recovered from a layer of a density of 1.090 g/ml. The isolated microfilariae retained their motility up to 3 days when cultured in vitro.