Treatment of co-infection with bancroftian filariasis and onchocerciasis: a safety and efficacy study of albendazole with ivermectin compared to treatment of single infection with bancroftian filariasis

BACKGROUND: In order to use a combination of ivermectin and albendazole for the elimination of lymphatic filariasis, it is important to assess the potential risk of increased adverse events in individuals infected with both lymphatic filariasis and onchocerciasis. We compared the safety and efficacy of albendazole (400 mg) in combination with ivermectin (150 micrograms/kg), for the treatment of co-infections of Wuchereria bancrofti and Onchocerca volvulus with single infection of W. bancrofti. METHODS: The safety study on co-infections was a crossover, double blind design, while for the single infection of bancroftian filariasis an open design comparing two treatments was used. For co-infection, one group was allocated a single dose of ivermectin (150 micrograms/kg) plus albendazole (400 mg) (Group A). The other group received placebo (Group B). Five days later the treatment regime was reversed, with the Group A receiving placebo and Group B receiving treatment. For the single bancroftian filariasis infection, one group received a single dose of albendazole (400 mg) plus ivermectin (150 μg/kg) (Group C) while the other group received a single dose of albendazole (400 mg) alone (Group D). Blood and skin specimens were collected on admission day, day 0, and on days 2, 3, and 7 to assess drug safety and efficacy. Thereafter, blood and skin specimens were collected during the 12 months follow up for the assessment of drug efficacy. Study individuals were clinically monitored every six hours during the first 48 hours following treatment, and routine clinical examinations were performed during the hospitalisation period and follow-up. RESULTS: In individuals co-infected with bancroftian filariasis and onchocerciasis, treatment with ivermectin and albendazole was safe and tolerable. Physiological indices showed no differences between groups with co-infection (W. bancrofti and O. volvulus) or single infection (W. bancrofti). The frequency of adverse events in co-infected individuals was 63% (5/8, Group A, albendazole + ivermectin) and 57% (4/7, Group B, placebo) and of mild or moderate intensity. In single W. bancrofti infection the frequency of adverse events was 50% (6/12, Group C, albendazole + ivermectin) and 38% (5/13, Group D, albendazole) and of a similar intensity to those experienced with co-infection. There were no differences in adverse events between treatment groups. There was no significant difference in the reduction of microfilaraemia following treatment with albendazole and ivermectin in groups with single or co-infection. CONCLUSION: Our findings suggest that ivermectin plus albendazole is a safe and tolerable treatment for co-infection of bancroftian filariasis and onchocerciasis

Production Of Silk Fibroin Scaffolds With Remarkable Mechanical Properties At High Fibroin Concentrations

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2016Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2016Dejeneratif, cerrahi ya da travmatik süreçlere bağlı olarak oluşan kemik kırıklarında veya kemik doku kayıplarında iyileşmeyi ve yenilemeyi sağlamak için doku isketleri kullanılmaktadır. Doku isketleri bu süreçte hücrelerin doğal yapılarına ulaşması için, destekleyici ve koruyucu olmalıdır. Ayrıca, toksik olmamalı, biyouyumlu ve biyobozunur olmalı en önemlisi kolayca üretilmelidir. Doku iskeletleri, sadece biyolojik değil aynı zamanda belirli kimyasal ve fiziksel özellikleri de sağlamalıdır. Bu nedenle mekanik fonksiyonlarını yerine getirecek sağlamlık ve sertliğe sahip olmalıdır. Birbiri ile bağlantılı gözenekli yapısı kontrol edilebilir ve gözeneklilik oranı % 90'nın üzerinde olmalıdır. Anlaşılacağı gibi, doku isketlerinde gözeneklilik ve mekanik özellikler arasındaki bu  yakın ilişki oldukça fazla öneme sahiptir. Hidrojellerin pratik uygulamalarında, dışarıdan gelen uyarılara hızlı tepki vermesi, aynı zamanda mekanik dayanımının çok yüksek olması amaçlanmaktadır. Ancak, hidrojellerin uygulamalarını sınırlayan en büyük etken mekanik olarak dayanıksız oluşlarıdır. Dayanıklı ve tok hidrojellerin eldesi için son yıllarda yoğun araştırmalar yapılmakta,  çeşitli teknikler geliştirilmekte, diğer yandan onlara  makrogözenekli bir yapı kazandırılması amacıyla  çalışmalar yapılmaktadır. Bu araştırmalar ışığında kriyojelleşme tekniği ile makro gözenekli ve mekanik özellikleri geliştirilmiş jellerin elde edilmesi mümkün olmaktadır. Kriyojelleşme, reaksiyon çözeltisinin donma noktasının altındaki uygun bir sıcaklıkta soğutularak oluşan çözücü kristallerinin etrafındaki donmamış mikro bölgelerde konsantrasyon artışına bağlı olarak ilerler. Kriyokonsantrasyon etkisi olarak adlandırılan bu durum, kriyojeleşmenin temel prensibidir. Elde edilen jelin (kriyojel) üstün özelliklerinin oluşmasında etkilidir. Donmuş çözücü ise, kalıp etkisi oluşturarak kriyojelleşme sonrasında eritilmesiyle, birbiri ile bağıntılı gözenekleri oluşturmasını sağlar. Kemik doku mühendisliğinde yapı iskeleti olarak kullanılması amacıyla, biyobozunur, biyouyumlu ve üstün mekanik özelliklere sahip olan ipekten yola çıkarak, laboratuarımızda kriyojeller üretilmiştir.  İpek yapısında bulunan fibroin proteini, başlıca glisin ve alanin amino asit ünitelerinden oluşan ve hidrofobik bloklar ile bunların aralarında hidrofilik bloklar içeren bir kopolimer yapısındadır. Hidrofilik bloklar suda çözünürlüğü sağlarken, hidrofobik bloklar arası asosiyasyonlar, fibroinin rastgele yumak yapısından β-tabaka yapısına bir konformasyon geçişine neden olur. İpek fibroinin (İF) yapısındaki β-tabakaları malzemeye dayanıklılık ve sertlik kazandırırken, daha düzensiz olan hidrofilik bloklar tokluğu ve elastisiteyi arttırır.  Tez kapsamında yapılan deneysel calışmalarda, gerek 1,4-bütandiol diglisidil eter (BDDE) çapraz bağlayıcısı ve gerekse sodyum dodesil sülfat (SDS) sürfaktanı kullanılarak fibroinin β-tabaka konformasyonuna geçişi sağlanmış ve kriyojeller elde edilmiştir.  BDDE çapraz bağlayıcı kullanılarak,fibroinin jelleşmesi -18oC' de, 24 saatte gerçekleştirilmiştir. % 1 ile % 61,4 arasında değişen fibroin konsantrasyonlarında hazırlanan kriyojellerin şişme özellikleri, konformasyonel değişimleri, mekanik özellikleri ve gözenekli yapısı açıklanmıştır. Fibroinin jelleşmesi sırasındaki konformasyonel değişimler ATR-FTIR ölçümleri ile belirtilmiştir. Kriyojelleşme ile fibroin rastgele yumak yapısından β-tabakaya geçişi gösterilmiştir. Şişme ölçümleri yapılmış kriyojellerin hacimce ilave şişmeyip, fakat kütlece şiştiği belirtilmiştir. Şişme ölçümleri sonucunda gözeneklilik oranının fibroin konsantrasyonuna bağlı olarak % 97 ile % 69 arasında ayarlanabileceği ortaya çıkmıştır. Farklı fibroin konsantrasyonlarında hazırlanan kriyojellerin kuru durumdaki mekanik özellikleri test edilmiştir. En çarpıcı sonuç, % 46,2 İF konsantrasyonunda hazırlanan kriyojelin Young modülünün  126 MPa değerine ulaşmasıdır.    Ayrıca, kriyojellerin parçalanma gerilimleri ve deformasyon oranları belirlenmiştir. Farklı konsantrasyonlarda hazırlanan tüm kriyojeller % 95-100' e kadar deforme olabilmektedirler. Kriyojellerin parçalanma gerilimleri 243 MPa kadar çıkmaktadır.  SEM görüntüleri incelenmiş, kriyojelleşme ile düzenli gözeneklerin oluştuğu görülmüştür. Gözenek boyutu ile mekanik özellikler arasındaki ilişki açıklanmıştır. Artan fibroin konsantrasyonuyla gözenek duvarlarının kalınlaştığı, gözenek boyutlarının küçüldüğü, kriyojellerin daha dayanıklı olduğu yorumlanmıştır. Kuru durumdaki ölçümlerine ek olarak, suda dengeye gelmiş kriyojellerin mekanik ölçümleri yapılmıştır. Kuru durumdakilere oranla, mekanik test sonuçlarının önemli ölçüde düştüğü saptanmıştır. Şişmiş kriyojellerin bu durumu, fibroin ağ yapı zincirlerinin hidrofilik yapısı nedeniyle gözenek suyunu tutması ve kauçuksu duruma geçmesi ile açıklanmıştır. SDS sürfaktanı kullanılarak, ipek fibroinin  jelleşmesi kriyojelleşme tekniği ile hızlandırılmıştır ve -18oC' de ve 48 saatte gerçekleştirilmiştir. SDS sürfaktanının fibroin zincirleri arasındaki hidrofobik etkileşimleri tetiklediği ve böylece jelleşmeyi sağlayan ajan olarak davrandığı düşünülmektedir. % 4,2 İF konsantrasyonunda 5 mM ile 125 mM arasındaki farklı SDS konsantrasyonlarında kriyojeller sentezlenmiştir. Sürfaktan miktarının, fibroinin kriyojelleşme prosesinde önemli bir farklılığa yol açmadığı gözlemlenmiştir.  Tüm SDS konsantrasyonlarında kriyojellerin şişme özellikleri benzer davranış göstermiştir ve gözeneklilik % 97 civarında hesaplanmıştır. Mekanik ölçümler sonucunda 5 mM SDS konsantrasyonunda elde edilen kriyojelin biraz daha sağlam olduğu gözlenmiştir. Düşük SDS konsantrasyonlarında fibroin zincirleri arasında oluşan hidrofobik blokların daha düzenli olduğu düşünülmüştür. SEM görüntüleri incelendiğinde 5 mM SDS konsantrasyonundaki kriyojelin gözenekli yapısının daha iyi olduğu görülmüş ve mekanik özellikler ile tutarlı olduğu saptanmıştır. Yapılan çalışmalar sonucunda, ipek fibroinin donmuş sulu çözeltilerinde  kriyojelleşmesi ile  makro gözenekli, hemen hemen % 100 deforme olabilen, mekanik özellikleri çok iyi kriyojeller elde edilmistir.  Bu tip malzemeler, kemik doku mühendisliği uygulamalarında yapı iskeleleri olarak kullanıma uygun iskeletlerdir.The deformation and degeneration of bone with age, trauma, congenital defects, and tumor cause not repairable damage to bone, resulting in increasing requirements for bone implants. Bone tissue engineering is a promising strategy to regenerate bone and is regarded as a future alternative to current clinical treatments. In this whole process, the patient's own cells could be used which would be a key tool for personalized medicine. The aim is to make 3D bone tissues by combining cells, scaffolds and to some extent also growth factors or mechanical stimuli. One of the main challenges is the choice of an appropriate biomaterial which can mimic the natural bone tissue matrix with its mechanical and biological characteristics to support tissue development. Various materials have been tested for bone tissue engineering purposes. An ideal scaffold for bone tissue engineering should be biocompatible, biodegradable with an architecture that mimics in vitro extra cellular matrix and possess good mechanical properties to facilitate bone formation. Pore structure also is an essential consideration in the development of scaffolds for tissue engineering. Pores must be interconnected to allow for cell growth, migration and nutrient flow. Regeneration of large bone defects is challenging in the next few years because the age of the population is growing. In recent years, many novel materials and processing methods have been introduced to construct the native bone extracellular matrix and restore the functions of degenerated bone. Silk fibroin as a natural polymer, has established a good reputation for bone tissue engineering applications due to its many unique properties. Silks are generally defined as protein polymers that are synthesized and spun into fibers by many organisms, including silkworms, spiders, scorpions, mites and flies. The more commonly utilized silk, such as from the silkworm, Bombyx Mori, has been used for thousand years due to its visual appeal and mechanical properties. The silk consist of fibroin held together with a layer of sericin on their surfaces. Upon degumming silk to remove the sericin, one obtains fibroin fibers for many applications.  Silk fibroin derived from Bombyx Mori is a fibrous protein exhibiting extraordinary material properties such as good biocompibility, biodegradabilitiy, high strength and toughness and also ease of processability. Silk fibroin has been used for cell culture, wound dressing, drug delivery and it  has been proven to be a promising biomaterial for scaffold fabrication in general and its remarkable mechanical properties appreciate it for bone tissue engineering applications. Silk fibroin has a blocky structure consisting of less ordered hydrophilic and crystallizable hydrophobic blocks. Hydrophilic blocks provide solubility in water and responsible for fibroin elasticity and toughness, while hydrophobic blocks form intermolecular β-sheet structures leading to the insolubility and high strenght of fibroin. One class of polymeric materials is hydrogels, which consist of three-dimensional solid networks made from crosslinked hydrophilic polymer chains swollen in water. Owing to their outstanding characteristics such as water absorption and retention ability, biocompatibility and tunable physical, chemical and biological properties, polymeric networks are excellent candidates for a broad range of biomedical applications, which include scaffolds for tissue engineering, carriers for drug delivery, molecular filters in biological science and superabsorbent devices.  Furthermore, when exposed to an environmental signal such as temperature and pH, some sensitive hydrogels are able to respond and translate this stimulus into a macroscopic event, allowing their application as sensors or actuators and smart drug release devices. But, the most interesting feature about these 3D networks is their high resemblance, in terms of structure and physical properties, with the native extracellular matrix, highlighting their potential for tissue engineering and biomedical purposes. However, their use in stress-bearing applications is often hindered since hydrogels, when highly swollen, often lack of mechanical properties such as strength, toughness, elongation and recoverability. This poor mechanical performance is in high contrast with native structural hydrogels such as cartilage, bone, tissue and may result in unintended failure in vivo. Hydrogels were classified as mechanical weak materials. Nevertheless, in the last few years, new strategies have been developed to toughen hydrogels including double networks, topological and nanocomposite hydrogels and also cryogels; allowing the application of these 3D structures, for instance, as artificial substitutes of native tissues with structural properties similar to hydrogels such as skin , heart valves, spinal disks , cartilage , muscles and nerves. In recent years, strategies for the synthesis of macroporous gels have been continually optimized and the concept of cryogelation has become increasingly important for the synthesis of such smart materials. Cryogelation is a simple strategy that allows the preparation of macroporous gels with high toughness and superfast responsivity. Cryogels are producted in frozen systems known as cryogelation. Cryogels are very tough gels that can withstand high levels of deformation, and can be squeezed almost completely without any crack propagation. Current research in the field of macroporous gels is focused on formation the pore structure. The crosslinking reactions at subzero temperatures that lead to cryogels are multicomponent systems composed of a polymer network with solvent crystals. All concentrations and properties of the system components change continuously during the cryogelation process. A more complete understanding of this complicated gel formation system is needed in order to improve the structuring of cryogels overall length scales from nanometer to micrometer. Moreover, because the volume swelling ratio of the cryogels and their porosity are inversely coupled, new synthetic approaches are also needed for the preparation of cryogels exhibiting drastic volume changes in response to external stimuli.  In cryogelation reaction solution, generally containing the monomer and the iniator, is cooling below the freezing point of the system. Since the monomers/polymers and the iniator will be enriched in unfrozen microzones surrounded by ice crystals. The cross-linking reactions only proceed in these unfrozen regions containing a high concentration of polymer the increased polymer concentration in the unfrozen reaction zones polymer concentration in the unfrozen reaction (cryoconcentration) and is responsible for extraordinary properties of cryogels. A macroporous structure appers due to the existence of solvent crystal acting as template or porogen for formation of pores. In contrast to the mechanically weak macroporous gels prepared by phase separation technique, cryogels are very tough and withstand very large strains without permanent deformation or fracture. Cryogel is an emerging class of biomaterials which have recently started as potential tissue scaffolds for regenerative medicine In this thesis, formation conditions and properties of mechanically strong macroporous cryogels based on silk fibroin were investigated.  Gelation reactions of silk fibroin were conducted at -18oC.  In the first part of this thesis, silk fibroin cryogels were synthesized at various fibroin concentrations in the presence of 1,4-butanediol diglycidyl ether (BDDE) as a cross-linker and N,N,N',N'-tetramethylethylenediamine (TEMED) as a catalyst.   Swelling behaviour, mechanical and morphological properties of the cryogels were investigated depending on the fibroin concentration in the gelation solution.  For this purpose, aqueous solutions of fibroin at various concentrations between 1 and 61.4 w/v % were mixed with BDDE and TEMED. The homogenous reaction solution was transferred into several plastic syringes of 4 mm internal diameters and then, they were placed in a thermostat at -18oC to conduct the cross-linking  reactions for 24 hours. Epoxide groups on the both ends of BDDE react  with the amino groups of fibroin to form crosslinks between fibroin molecules. As reported before, BDDE  triggers the conformational transition of fibroin from random coil to β-sheet structure and hence, fibroin gelation. The swelling equilibrium was tested by measuring the diameter of the gel samples by using digital compass and also the swelling equilibrium was tested by weighing  the gel samples. Then, the equilibrium swollen gel samples were frozen in a freeze-drying system to obtain dry cryogels. While the equilibrium weight ratio qw decreases from 35 to 3, the equilibrium volume ratio qv   does not change much and remains around unity with increasing silk fibroin concentration. We estimated the total volume of the pores in the cryogel scaffolds from their weight qw and volume swelling ratios qv. Because the weight swelling ratio includes water locating in both pores and in the fibroin region of the gel, while, assuming isotropic swelling, the volume swelling only includes water in the fibroin gel, the larger the difference between qw and qv, the larger the amount of water in the pores, that is, the larger the volume of pores in the scaffolds. From the weight and volume swelling ratios of the cryogels, the porosity P was estimated. The porosity of cryogel scaffolds decreases from 97 to 69 % with increasing fibroin concentration from 1 to 61.4 %. Moreover, measurements also show that the gel fraction Wg ≅ 1 at fibroin concentrations between 4 and 30 %  while at higher  fibroin concentrations, it becomes almost equal to 0.5. The conformation of fibroin molecules in the cryogels  was assessed by ATR-FTIR spectra of freezed-dried cryogel samples. The spectrum of fibroin before gelation is characterized by a peak at 1640 cm−1 indicating the presence of primarily random coil and/or α- helix conformations. After cryogelation, all samples display a main peak at 1620 cm−1 which was assigned to β-sheet conformation. This indicates the occurrence of a conformational transition from random coil to β-sheet structure during the cryogelation reactions.  It was also shown that the scaffolds of fibroin cryogels consist of  interconnected pores. The pore size of the cryogel scaffold could be regulated depending on the fibroin concentration. Increasing fibroin concentration led to scaffolds with thicker pore walls, but smaller pore size. The pore diameter decreased from 28 ± 7 to 9 ± 4 μm as silk fibroin concentration increased from 4.2 to 61.4 %. Mechanical properties of the scaffolds were investigated by uniaxial compression tests. The modulus and strength of cryogel networks changed depending on the fibroin concentration at the gel preparation. The compressive modulus E increases from 7 ± 2 to 126 ± 2 MPa with increasing fibroin concentration from 4.2 to 46.2 %.  Simultaneously, the plateau and compressive stresses also increase from 0.3 to 7.6 MPa and from 0.1 to 13 MPa, respectively, with increasing fibroin concentration.   Comparison of the results of the mechanical tests with the morphology of cryogels  clearly show that a decrease in the average pore diameter increases the mechanical stability of fibroin scaffolds formed by cryogelation. Furthermore, fracture stress of cryogels scaffolds also increases from 16 ± 2 to 243 ± 23 MPa  with increasing fibroin concentration from 4.2 to 11.6 %. Above 11.6 % fibroin concentration, the fracture stress does not change much with increasing silk fibroin concentration. All the fibroin cryogels reported in this thesis  remained mechanically stable without any fracture up to 95-100 % compressions. In the second part of the thesis, instead of BDDE and TEMED,  sodium dodecyl sulfate (SDS) was used to trigger conformational transition in fibroin and hence,  fibroin gelation. The gelation reactions were carried out at a fibroin concentration of 4.2 w/v % in the presence of SDS at various concentrations between 5 and 125 mM. Homogenous reaction solution containing fibroin and SDS was transferred into several plastic syringes of 4 mm internal diameters and then, they were placed in a thermostat at -18oC to conduct gelation reactions for 2 days.  It was found that  gelation of fibroin using SDS occurs faster than using BDDE crosslinker. We may speculate that the alkyl chains of SDS bring hydrophobic blocks of fibroin together due to hydrophobic interactions which accelerates conformational transition in fibroin chains. The conformational change in fibroin due to the SDS-induced gelation was assessed by ATR-FTIR spectra of freezed-dried fibroin samples. The spectrum of fibroin before gelation is characterized by a peak at 1640 cm−1 indicating the presence of primarily random coil and/or α- helix conformations. After cryogelation, all samples display a main peak at 1620 cm−1 which was assigned to β-sheet conformation. This indicates the occurrence of a conformational transition from random coil to β-sheet structure in frozen fibroin solutions. We have to mention that   cryogelation and thus, gelation do not occur without SDS in gelation solution. All fibroin scaffolds formed at various SDS concentration (from 5 to 125 mM) exhibit similar swelling behavior. The equilibrium weight qw and equilibrium volume swelling ratios qv  were measured as 35 and 1, respectively, independent of the SDS concentration.   In addition, porosity of all  scaffolds was calculated as 97 % SEM images of  fibroin scaffolds formed in presence of SDS showed that cryogels consist of more regular pores at low surfactant concentration. Moreover, the scaffolds formed at 5 mM SDS exhibited a  compressive modulus E  of 1.0 MPa, while  it reduced to 0.2 MPa when SDS concentration is increased to 125 mM SDS. The results thus show that, in the presence of BDDE crosslinker, silk fibroin cryogels with remarkable properties were obtained from frozen fibroin solutions (4.2−61.4%) at −18 °C. One of the unique features of fibroin cryogels is their elasticity that allows them to resist up to 95-100 % compression without any crack development. The scaffolds obtained by freeze-drying of the cryogels consist of regular, interconnected macropores of diameters ranging from 28 to 9 μm that could be regulated by the fibroin concentration. The mechanical compressive strength and the modulus of the scaffolds increase with decreasing pore diameter, that is, with increasing fibroin content. Especially, the scaffolds produced at 46.2 % fibroin exhibit a very high compressive modulus (126 MPa) making them good candidates as bone scaffold materials.Yüksek LisansM.Sc

Onchocerciasis-associated epilepsy: another piece in the puzzle from the Mahenge mountains, southern Tanzania

Multilingual abstracts in the five official working languages of the United Nations. (PDF 488 kb