Synthesis of some 3,4-disubstituted-6,7-dihydro-imidazo[2,1-b][1,3]thiazole and 3,4-disubstituted-7,8-dihydro-6H-imidazo[2,1-b][1,3]thiazine derivatives and evaluation of their cytotoxicities against F2408 and 5RP7 cells

This article describes the synthesis of 3,4-disubstituted-6,7-dihydro-imidazo[2,1-b][1,3]thiazoles and 3,4-disubstituted-7,8-dihydro-6H-imidazo[2,1-b][1,3]thiazines, having substituted or nonsubstituted phenyl rings at the 5,6 and 2,3 positions, respectively, their cytotoxic effects through noncancer (F2408) and cancer (5RP7) cells, and their detailed ¹H- and ¹³C-nuclear magnetic resonance (NMR) spectral characterization. The title compounds were obtained by the cyclization of 4,5-diaryl-imidazole-2-thione and dihaloalkane (i.e., 1,2-dihaloethane or 1,3-dihalopropane), in the presence of potassium carbonate (K₂CO₃) in N,N-dimethyl formamide (DMF). 4,5-Diaryl-imidazole-2-thione was prepared by condensation of α-hydroxyketones (acyloins), which were obtained by treating aldehydes with cyanide, with thioureas in AcOH. The structure of imidazo[2,1-b][1,3]thiazole and imidazo[2,1-b][1,3]thiazine derivatives was confirmed by infrared (IR), ¹H-NMR, and ¹³C-NMR. The cytotoxicities of the synthesized compounds on both of noncancer (F2408) and cancer (5RP7) cells were measured by 3-(4,5-dimethyl-thiazollyl-2)-2,5-diphenyltetrazolium (MTT) assay. In the presence of only lower doses of compounds 9 and 11, bearing methyl or methoxy substituents on the phenyl ring of imidazo[2,1-b][1,3]thiazole scaffold, the cytotoxic effect was higher on 5RP7 cells than control cells after 24 h.     

Successful Liver-Kidney Transplantation in Two Children With aHUS Caused by a Mutation in Complement Factor H

A 12-month-old boy and his 16-year-old aunt became acutely ill 6 months apart and were diagnosed to have atypical hemolytic uremic syndrome (aHUS). Genetic analysis revealed heterozygous R1215Q mutation in complement factor H (CFH) in both patients. The same mutation was found in five healthy adult relatives indicating incomplete penetrance of the disease. The patients developed terminal renal failure and experienced reversible neurological symptoms in spite of plasma exchange (PE) therapy. In both cases, liver-kidney transplantation was successfully performed 6 months after the onset of the disease. To minimize complement activation and prevent thrombotic microangiopathy or overt thrombotic events due to the malfunctioning CFH, extensive PE with fresh frozen plasma was performed pre- and perioperatively and anticoagulation was started a few hours after the operation. No circulatory complications appeared and all four grafts started to function immediately. Also, no recurrence or other major clinical setbacks have appeared during the postoperative follow-up (15 and 9 months) and the grafts show excellent function. While more experience is needed, it seems that liver-kidney transplantation combined with pre- and perioperative PE is a rational option in the management of patients with aHUS caused by CFH mutation.     

Effects of amlodipine and fosinopril on heart rate variability and left ventricular mass in mild-to-moderate essential hypertension

The differences between long-acting dihydropyridines and angiotensin-converting enzyme inhibitors with regard to their long-term effects on 24-h heart rate variability (HRV) and left ventricular (LV) mass are less clear in mild-to-moderate essential hypertension. We studied the long-term effects of amlodipine and fosinopril on 24-h HRV and LV mass in mild-to-moderate essential hypertension. In this study, 27 patients with never treated mild-to-moderate essential hypertension were randomised to receive either amlodipine or fosinopril once daily as monotherapy. At baseline and at the end of the third and sixth months, each of the patients underwent 24-h HRV and ambulatory systolic (SBP) and diastolic (DBP) blood pressure analysis. LV mass index was calculated from echocardiographic examination at baseline and at the end of the sixth month. In amlodipine group (n = 14), 24-h SBP/DBP (mmHg) decreased from 144 +/- 8/94 +/- 4 to 128 +/- 6/83 +/- 3 at the end of the third month and to 125 +/- 5/81 +/- 2 at the end of the sixth month (p < 0.0001). In fosinopril group (n = 13), the respective changes were 143 +/- 9/97 +/- 7, 132 +/- 6/87 +/- 5 and 127 +/- 6/82 +/- 3 (p < 0.0001). At the end of the sixth month, LV mass index (g/m(2)) decreased from 122 +/- 26 to 105 +/- 21 in amlodipine group (p < 0.0001) and from 118 +/- 23 to 101 +/- 14 in fosinopril group (p < 0.0001). There were no significant changes in HRV parameters in both the groups. It was concluded that both drugs caused significant decrease in SBP and DBP, and LV mass in patients with mild-to-moderate essential hypertension did not have significant long-term effects of either amlodipine or fosinopril on 24-h HRV parameters reflecting sympathetic or parasympathetic activity in these patients.     

Association of time-to-surgery with outcomes in clinical stage I-II pancreatic adenocarcinoma treated with upfront surgery

Background

Time-to-surgery from cancer diagnosis has increased in the United States. We aimed to determine the association between time-to-surgery and oncologic outcomes in patients with resectable pancreatic ductal adenocarcinoma undergoing upfront surgery.

Differential detection of nuclear envelope autoantibodies in primary biliary cirrhosis using routine and alternative methods

Background  

Detection of autoantibodies giving nuclear rim pattern by immunofluorescence (anti-nuclear envelope antibodies - ANEA) in sera from patients with primary biliary cirrhosis (PBC) is a useful tool for the diagnosis and prognosis of the disease. Differences in the prevalence of ANEA in PBC sera so far reported have been attributed to the methodology used for the detection as well as to ethnic/geographical variations. Therefore, we evaluated the prevalence of ANEA in sera of Greek patients with PBC by using methods widely used by clinical laboratories and a combination of techniques and materials.     

Fish intake and LPA 93C>T polymorphism: gene-environment interaction in modulating lipoprotein (a) concentrations

High plasma lipoprotein (a) [Lp(a)] concentrations are an independent risk factor for atherosclerotic diseases. To date, no effective intervention strategies on reducing Lp(a) concentrations have been reported. The aim of the study was to evaluate the possible modulation of two polymorphisms of LPA gene (LPA 93C>T and LPA 121G>A) and nutritional habits on Lp(a) concentrations. We studied 647 healthy Italian subjects (260 M; 387 F) with a median age of 48 years (range: 19-78) enrolled in an epidemiological study conducted in Florence, Italy. A linear regression analysis showed a significant negative influence of fish intake (beta=-0.174+/-0.084; p=0.04) on Lp(a) concentrations, after adjustment for smoking habit, C-reactive protein serum concentrations, dietary habits and LDL-cholesterol concentrations. With regard to LPA polymorphisms, LPA 93C>T polymorphism resulted to significantly affect Lp(a) circulating concentrations in a dose-dependent manner, with lower concentrations shown by subjects carrying the T rare allele, whereas no significant influence of LPA 121G>A polymorphism on Lp(a) concentrations was observed. Moreover, by analyzing the possible interplay between LPA 93C>T and dietary fish intake, a significant interaction between these two determinants in lowering Lp(a) concentrations was reported. In addition, lower Lp(a) concentrations were observed in subjects carrying the T allele of the LPA 93C>T polymorphism and consuming a high intake of fish with respect to those being in the highest tertile of fish consumption but homozygotes for the common allele of the polymorphism. In conclusion, this study reported a significant interaction of daily fish intake and LPA 93C>T polymorphism in decreasing Lp(a) concentrations.     

Loa loa Microfilariae Evade Complement Attack In Vivo by Acquiring Regulatory Proteins from Host Plasma

Loa loa is a filarial nematode that infects humans. The adults live in subcutaneous tissues and produce microfilariae that live for several weeks in the blood circulation in order to be transmitted to another person via blood meals of a dipterian vector. As microfilariae live in continuous contact with plasma, it is obvious that they evade the complement system. We studied markers of complement activation and signs of complement regulation on Loa loa microfilariae in vivo. The microfilariae were isolated from anticoagulated blood samples of a Loa loa-infected Caucasian patient. C1q and some mannose-binding lectin but only a limited amount of C3b or C4b fragments and practically no C5 or C5b-9 were present on the microfilariae. The covalently microfilaria-bound C3 and C4 depositions were mainly inactive iC3b, C3c, and iC4b fragments indicating that microfilariae had regulated complement activation in vivo. Also, in vitro deposition of C3b onto the microfilariae upon serum exposure was limited. The patient-isolated microfilariae were found to carry the host complement regulators factor H and C4b-binding protein on the outermost layer, so called sheath. The microfilaria-bound factor H was functionally active. Binding of the complement regulators to the microfilariae was confirmed in vitro using ¹²⁵I-labeled factor H and C4b-binding protein. In conclusion, our study shows that Loa loa microfilariae block complement activation and acquire the host complement regulators factor H and C4b-binding protein in blood circulation. This is the first time that binding of complement regulators onto nonviral pathogens has been demonstrated to occur in humans in vivo.Loa loa is a filarial parasite and the causative agent of human loiasis. This nematode has adapted well to its human host, as the adults can migrate in subcutaneous tissues for at least 15 years (15). During its whole adult life this helminth can produce microfilariae (MF) in peripheral blood, and these can circulate in blood for several weeks before transmission to the vector. Loiasis is transmitted by a dipteran vector (Chrysops spp.). The infective larvae can enter human subcutaneous tissues through a bite wound when the vector feeds again after the first blood meal. The larvae develop into adults and mate in subcutaneous tissues, resulting in production of sheathed MF after a minimum of 5 months. The MF are found mainly in peripheral blood (34).The prevalence of loiasis is high in the regions of endemicity in western and central Africa, where 20 to 40% of the population is microfilaremic (10). The majority of the infected individuals are asymptomatic, but a significant proportion of patients have symptoms such as calabar swellings, pruritis, secondary dermal lesions, and a subconjunctival eye passage of the adult worm (33, 37). In addition, serious sequelae such as endomyocardial fibrosis, renal complications, and encephalitis have also been reported (1).The main functions of the complement system are to eliminate foreign organisms that come in contact with plasma or other body fluids, either by direct effects or by enhancement of the acquired humoral immune response. Depending on the activator, complement can be activated through three pathways: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). Upon activation, these pathways lead to the terminal pathway and formation of membrane attack complexes on the target cell (31).Complement activation is regulated by a variety of complement regulatory proteins. Most of the regulators are membrane bound, while two major regulators, complement factor H (CFH) and C4b-binding protein (C4BP), are plasma proteins found in high concentrations (12, 17, 47). All the three pathways lead to activation of C3, the central molecule of the complement cascade (8). Activation of C3 to C3b results in release of an anaphylatoxin, C3a (20, 21), while the C3b fragment can attach covalently to the target surface to start the AP amplification or to promote activation of the CP or LP (32). Inactivation of C3b to inactive C3b (iC3b) is carried out by serine protease factor I (FI), which needs a cofactor such as CFH (6, 8). In addition to the cofactor activity, CFH can also downregulate generation of C3b by two other means (11, 46).In the CP and LP, activation of the component C4 is essential. Upon activation, C4b is attached covalently to the target surface (27, 45) and can form an active C3-convertase, C4b2a, which is essential for propagation of the CP and LP (5). This step is regulated in plasma by complement regulatory protein C4BP, which acts as a cofactor for FI in degradation of C4b to inactive C4b (iC4b) or as an accelerator of the decay of C4b2a (23, 40, 44).Since MF of Loa loa are able to live and migrate in blood for weeks, they are obviously able to resist elimination by complement, but the mechanisms are largely unknown. One immune evasion mechanism of Loa loa is known to be induction of T-cell anergy (25), but nothing is known about evasion of innate or humoral immunity. A few complement resistance mechanisms have been reported for other helminths. Most of these are associated with physical barriers of macroscopic worms, but some helminths are known to have specific molecules mediating complement evasion or ligands that acquire host complement regulators on their surfaces (22). So far, two human helminth parasite structures have been shown to acquire host CFH on their surfaces, the echinococcal cyst wall and MF of a filarial nematode, Onchocerca volvulus (7, 30). So far there are no reports of acquisition of C4BP onto any pathogenic helminth. Several pathogenic bacteria and yeasts and a few viruses are known to utilize acquisition of host CFH or C4BP to evade complement attack (49). Four of these microbes, Streptococcus pyogenes, Borrelia burgdorferi, relapsing fever Borrelia, and Candida albicans, can acquire both CFH and C4BP on their surface (3, 28, 29). So far, all the reports where CFH or C4BP acquisition on microbes has been reported have been based on in vitro experiments only.The aim of our study was to analyze whether patient-derived Loa loa MF carry any markers of complement attack or signs of cessation of the complement cascade. The MF showed C1q deposits, as was expected since the patient had antifilarial antibodies. Despite that, however, only limited amounts of C3 or C4 fragments and practically no C5 or C5b-9 could be detected on MF. The covalently bound C3 or C4 fragments were mainly iC3b, C3c, or iC4b. Most importantly we show that MF had acquired CFH and C4BP on their surfaces in vivo. In conclusion, for the first time we show acquisition of soluble complement regulators on pathogenic microbes in the human body. Our results suggest that acquisition of complement regulators CFH and C4BP from human plasma on MF could at least partially explain the prolonged survival of MF in circulation.     

Streptococcus pneumoniae Capsular Serotype 19F Is More Resistant to C3 Deposition and Less Sensitive to Opsonophagocytosis than Serotype 6B

The polysaccharide capsule is a major virulence mechanism of Streptococcus pneumoniae, shielding the bacterium from phagocytes. Capsule types may differ in their abilities to resist immune defense. Antibody-mediated complement activation and opsonophagocytosis are crucial in protection against pneumococcus. Conjugate vaccine trials suggest imperfect protection against 19F. We have previously shown that significantly more anti-19F than anti-6B antibody is needed for killing in the opsonophagocytic assay (OPA). In this study, we explored whether the amount of C3 deposited on serotype 6B and 19F pneumococcal strains reflects their sensitivity to opsonophagocytosis. We compared clinical 6B and 19F nasopharyngeal, middle ear, and blood isolates as well as reference OPA strains (n = 16) for their sensitivity to opsonophagocytosis and C3 deposition. Sixfold anticapsular antibody concentrations were required for 50% opsonophagocytic killing of 19F compared to that of 6B strains. Serotype 19F was more resistant to C3 deposition than 6B. Complement deposition and opsonophagocytosis were dependent on the concentration of anticapsular antibodies. Differences between pneumococcal serotypes in antibody-mediated protection may partly be explained by the abilities of the capsules to resist complement deposition. These findings support previous studies suggesting that higher antibody concentrations to the capsular polysaccharide are needed for protection against disease caused by serotype 19F than that caused by 6B.