Uptake of diethylcarbamazine by microfilariae and adults of Litomosoides carinii and Dipetalonema viteae

Microfilariae of Litomosoides carinii and Dipetalonema viteae absorbed about ten times as much diethylcarbamazine (DEC) as did their adults, but bound much less. The higher uptake of DEC by the microfilariae might be related to the fact that DEC is microfilaricidal. Binding of DEC with the parasites supports the view that the microfilaricidal action begins with the fixation of the drug to the microfilariae.     

Occurrence of microfilariae in various hollow organs and in the peritoneal cavity after treatment of Litomosoides carinii infected Mastomys natalensis with diethylcarbamazine and haloxon

Treatment of Litomosoides carinii infected Mastomys natalensis with diethylcarbamazine (DEC: 500 mg/kg p.o.) was followed by increased occurrence of microfilariae in the bronchi of the host after 40 min and lasting at least until 6 h after treatment. After 4 h, increased levels of larvae were observed in the gut. Only a few microfilariae occurred in the bladder and sputum. Accumulations of microfilariae were found furthermore in the Lnn. hepaticae whereas no changes were observed in the inguinal or jejunal and lung and pleura associated lymph nodes. Increased numbers of microfilariae were found in the peritoneal cavity only after 8 and continuing until at least 48 h after treatment. In contrast, after haloxon treatment (100 mg/kg p.o.) an accumulation of microfilariae was found in the peritoneal cavity only, following a time course similar to that after DEC.     

Diethylcarbamazine activity against Brugia malayi microfilariae is dependent on inducible nitric-oxide synthase and the cyclooxygenase pathway

Background

Diethylcarbamazine (DEC) has been used for many years in the treatment of human lymphatic filariasis. Its mode of action is not well understood, but it is known to interact with the arachidonic acid pathway. Here we have investigated the contribution of the nitric oxide and cyclooxygenase (COX) pathways to the activity of DEC against B. malayi microfilariae in mice.

Methods

B. malayi microfilariae were injected intravenously into mice and parasitaemia was measured 24 hours later. DEC was then administered to BALB/c mice with and without pre-treatment with indomethacin or dexamethasone and the parasitaemia monitored. To investigate a role for inducible nitric oxide in DEC's activity, DEC and ivermectin were administered to microfilaraemic iNOS^-/- mice and their background strain (129/SV). Western blot analysis was used to determine any effect of DEC on the production of COX and inducible nitric-oxide synthase (iNOS) proteins.

Results

DEC administered alone to BALB/c mice resulted in a rapid and profound reduction in circulating microfilariae within five minutes of treatment. Microfilarial levels began to recover after 24 hours and returned to near pre-treatment levels two weeks later, suggesting that the sequestration of microfilariae occurs independently of parasite killing. Pre-treatment of animals with dexamethasone or indomethacin reduced DEC's efficacy by almost 90% or 56%, respectively, supporting a role for the arachidonic acid and cyclooxygenase pathways in vivo. Furthermore, experiments showed that treatment with DEC results in a reduction in the amount of COX-1 protein in peritoneal exudate cells. Additionally, in iNOS^-/- mice infected with B. malayi microfilariae, DEC showed no activity, whereas the efficacy of another antifilarial drug, ivermectin, was unaffected.

Conclusion

These results confirm the important role of the arachidonic acid metabolic pathway in DEC's mechanism of action in vivo and show that in addition to its effects on the 5-lipoxygenase pathway, it targets the cyclooxygenase pathway and COX-1. Moreover, we show for the first time that inducible nitric oxide is essential for the rapid sequestration of microfilariae by DEC.     

Studies with Brugia pahangi 17. The anthelmintic effects of diethylcarbamazine.

Diethylcarbamazine (DEC) was active in vitro against infective larvae and microfilariae of Brugia pahangi but only at high concentrations. When fed to mosquitoes which were infected with B. pahangi it had little or no activity. In jirds it was inactive against B. pahangi microfilariae and adults when administered at 300 mg/kg for 5 days either by the intraperitoneal or oral route. In cats given 25 or 50 mg DEC/kg intraperitoneally on 3 or 5 occasions it was not microfilaricidal, but most of the adult worms died within 30 days of the end of treatment. Although most microfilariae disappeared from the blood of cats immediately (i.e., within an hour) after treatment, they reappeared within a few hours in the same numbers. Microfilarial levels were reduced after treatment but there was no precipitate decline as occurs in human B. malayi patients.     

Ultrastructural aspects of the degeneration of the dermal microfilaria O. volvulus under the effect of diethylcarbamazine in human onchocerciasis]

Early modifications observed by previous authors are confirmed. More over, some later modifications are described where microfilariae are completely destroyed and removed by phagocytic cells. The theory concerning the mode of action of DEC on microfilariae in vivo is not confirmed (absence of extracellular "coarse particulate material", which is actually the unique morphological finding which leads the previous authors to assess the presence of immune complexes at the surface of microfilariae).     

Studies on the mechanism of the DEC reaction in dogs infected with Dirofilaria immitis

Studies have been undertaken to investigate a number of potential mediators of canine hepatic vein constriction, the key event in the DEC shock reaction in dogs infected with Dirofilaria immitis. Using an isolated canine hepatic vein preparation it has been shown that α-adrenergic, H1 histaminic and D tryptaminergic receptors are present. There is also evidence that M tryptaminergic receptors are located in this tissue. Bradykinin does not contract the isolated hepatic vein, while DEC in high concentrations causes a dose-dependent contraction which is not mediated through its known ability to release noradrenaline. In vivo experiments using pharmacological antagonists have confirmed that histamine, 5-hydroxytryptamine, noradrenaline and adrenaline are not involved in this reaction. The reaction was not blocked by the endorphin antagonists naloxone and meptazinol. Dexamethasone, but not indomethacin however did block the reaction. Both anti-inflammatory agents act on biosynthetic pathways from arachadonic acid leading to the formation of leukotrienes and prostaglandins, respectively. However, since the dog is insensitive to leukotrienes it seems unlikely that these autocoids mediate the reaction. Therefore, it is postulated that the reaction results from the release, by DEC, of substances from microfilariae which are able to contract the hepatic veins either directly or indirectly and so induce hepatic venous congestion and hypovolaemic shock.     

Differential effects of diethylcarbamazine, tetracycline and the combination on Brugia pahangi adult females in vitro

Anti-filarial effects of diethylcarbamazine (DEC), tetracycline (TC) and the combination on Brugia pahangi adult females were studied in 7-day cell-free culture, in terms of microfilaria release, parasite motility, MTT assay for parasite viability and embryogram. TC 50 μg/ml (TC50) effectively reduced microfilaria release from day 1 of culture. Combined with DEC 100 μg/ml (DEC100) or DEC 500 μg/ml (DEC500), microfilaria release reduced further and synergistically. TC50 also reduced motility, but DEC100 and DEC500 did not. The combination of TC50 and DEC500 reduced motility synergistically. The MTT assay supported the results of motility study in general. The embryogram showed that only DEC500 reduced the total number of intrauterine embryos, especially ova, indicating that DEC500 inhibited early embryogenesis. TC50 did not affect the total number of embryos, but resulted in apparent accumulation of microfilariae in the uterus, suggesting that the drug inhibited release of microfilariae in this in vitro system. These results clarified different anti-female mechanisms between DEC and TC. A PCR-based study showed that endosymbiont bacteria, Wolbachia, in B. pahangi females decreased significantly after TC treatment. However, this study could not determine whether the effects of TC were direct or Wolbachia-mediated.     

Adverse drug reactions in the treatment of filarial parasites: Haematological, biochemical, immunological and pharmacological changes in Dirofilaria immitis infected dogs treated with diethylcarbamazine

In order to understand the mechanism of the shock reaction that can occur in Dirofilaria immitis-infected dogs, following treatment with diethylcarbamazine (DEC), a variety of blood parameters were measured. A decrease in red blood cell parameters occurred in reactive dogs, followed by an increase, suggesting a possible sequestration of erythrocytes followed by peripheral vasoconstriction. Changes in leucocytes were consistent with stress reactions. Thrombocytopenia occurred, but no evidence of coagulopathy was seen. Alanine aminotransferase and creatinine phosphokinase (especially CK1 and CK3) concentrations increased suggesting cell damage and possibly liver cell necrosis, possibly due to hypoxia. Complement was not activated during the reaction and there was no increase in circulating immune complexes. No changes in plasma concentrations or histamine, 5-hydroxytryptamine, kininogen or prostaglandin F metabolite were found. Bradycardia was not caused by changes in potassium or calcium ion concentrations. These results suggest that the shock reaction is not an anaphylactic Type 1 reaction and is not caused by mast cell degranulation or by immune complex activation of mediators. The most likely cause of the reaction is that DEC causes the liberation of substances from microfilariae or adult worms which are able to constrict the hepatic vein directly or via its sympathetic innervation.     

Binding properties of diethylcarbamazine

Diethylcarbamazine (DEC) reacted with liver cell plasma membrane of rodent hosts-cotton rat, albino rat and Mastomys natalensis exhibiting the presence of both saturable and unsaturable components. The presence of lectins or sugar derivatives did not affect the binding significantly. The drug showed similar binding pattern with serum but the saturation was reached at a much lower concentration of the ligand. Data obtained with a variety of macromolecules, particularly with the homopolymers of amino acids indicate that DEC does not require any specific constituent of the membrane for binding. The nonspecific nature of DEC binding does not provide any convincing clue for the accumulation of microfilariae specifically in the liver following the drug treatment.     

The effect of diethylcarbamazine on microfilariae of Litomosoides carinii in vitro and in vivo

Culture-derived Litomosoides carinii microfilariae (MFF) were used in in vitro and in vivo systems to investigate the effect of diethylcarbamazine (DEC) on these MFF. In vivo: Male rats, Mastomys natalensis, all of the same age, were injected intrathoracically (12) or intraperitoneally (36) with 10(3) or 10(4) MFF. After 30 min one half of each group of rats was given DEC per os. At 30, 60, and 120 min after DEC administration, two rats from the treated and two from the untreated group were bled and killed. The pleural or peritoneal cavities were rinsed with warm saline (0.15 M NaCl) to recover MFF. In both the intrathoracic and intraperitoneal experiments, equal numbers of MFF were recovered from treated and control rats at 30 and 120 min. However, at 60 min 85.5% fewer were recovered from the treated than from the nontreated animals. MFF were not found in the blood. In vitro: MFF were added to tissue culture dish wells (Linbro Div., Flow Labs, Hamden, Conn) prepared as follows: DEC-Serum (serum from normal rats given DEC at 500 mg/kg), DEC + Serum (serum with added DEC), serum only, RPMI 1640 only, and RPMI 1640 + DEC. Furthermore, the five treatments were prepared either with or without unstimulated peritoneal exudate (PE) cells. At 30 min in the DEC-Serum wells 45% of the MFF had adherent PE cells; in the remaining wells these cells adhered to 11% or fewer MFF. We interpret the aforementioned phenomena as representing the first step in the trapping and elimination of MFF after DEC treatment of L. carinii-infected M. natalensis.     

Comparative study of the cutaneous and ocular lesions of murids Lemniscomys striatus parasitized by Monanema spp. and Atherurus africanus parasitized by Cercopithifilaria sp. with those of human onchocercosis

Histologic examination of skin and ocular lesions in various rodents infested by various species of Filariae with skin-dwelling microfilariae disclosed very similar aspects as compared to lesions noted in human onchocerciasis: coexistence in the same organ of five following types of lesion: vascular changes; subacute inflammatory cells infiltration; granulomatous lesions; scarring alterations; vascular ectasia.     

Brugia malayi microfilaraemia in mice: a model for the study of the host response to microfilariae

Microfilariae of Brugia malayi were obtained from the peritoneal cavities of infected gerbils and were then injected intravenously into mice. A sub-periodic, nocturnal microfilaraemia was produced. The level of microfilaraemia was proportional to the number of parasites injected, with approximately 1-3% of microfilariae being found in the peripheral circulation. The duration of microfilaraemia was proportional to the number of parasites injected; it subsided by 30 days after injection of 104 microfilariae but was still present at a low level 120 days after injection of 2 x 105 microfilariae. A transient splenomegaly developed after injection of microfilariae. Histopathological examination revealed large numbers of microfilariae free in the lumens of pulmonary small blood vessels and without any accompanying inflammatory reaction. Lesser numbers of microfilariae were seen in the cardiac blood and hepatic and renal blood vessels for the first few days after injection. There was cellular proliferation in the splenic white pulp and vascular congestion of the red pulp. Microfilariae labelled with 51Cr were injected intravenously; 57% of radioactivity was found in the lungs, 8.5% in the liver and 2.9% in the spleen. Mice developed immediate hypersensitivity reactions to B. malayi antigen by 4 weeks after injection, but Arthus and delayed hypersensitivity reactions were not seen at any time. when mice which had been injected 5 months previously were challenged with a 2nd injection of microfilariae, there was an accelerated clearance of parasites over 2 weeks and a marked peripheral blood eosinophilia developed. In contrast with natural infections, in which the continuous production of microfilariae complicates assessment, this model provides a system in which factors controlling the circulation of microfilariae in the bloodstream can be studied independently.     

Microfilariae in association with other diseases. A report of six cases

BACKGROUND: Lymphatic filariasis is a major public health problem in tropical countries. Earlier reports have reported microfilariae as an incidental finding in body fluids and fine needle aspiration smears from various sites. CASES: The findings of body fluid cytology and fine needle aspiration smears from six patients with microfilariae in association with other conditions--tubercular pleural effusion/lymphadenitis, pregnancy and non-Hodgkin's lymphoma--are presented. Three patients demonstrated an associated eosinophilic cellular exudate. Adherence of inflammatory cells to microfilariae was seen in two patients. CONCLUSION: Although microfilariae in cytologic smears are considered incidental findings, the association of microfilariae with debilitating conditions suggests that it is an opportunistic infection and needs further study.     

DEC1-mRNA在肝癌患者血清中的表达和手术预后的关系

目的:研究DEC1在肝癌(hepatocellular carcinoma,HCC)患者血清中的表达水平及其临床意义。方法:收集60例HCC患者,30例正常人,30例慢性肝炎患者,30例慢性肝硬化患者血清标本。采用实时荧光定量RT-PCR(RT-PCR)检测血清DEC1的表达情况。分析血清DEC1的表达水平与AFP相关性。探讨血清DEC1的表达水平与HCC手术预后的关系。结果:肝癌患者血清DEC1明显高于正常人、慢性肝炎和慢性肝硬化患者(P0.05)。皮尔森相关分析结果显示HCC患者血清DEC1的表达与血清AFP水平是呈正相关的。Kaplan-Meier结果显示血清DEC1低表达组术后的复发转移率显著低于高表达组,其术后生存率也显著高于高表达组(P0.01)。结论:DEC1在肝癌者血清中高表达,不仅可能成为HCC诊断的标记物,且其血清表达水平也有助于HCC临床预后的评估。     

Red blood cell antioxidant levels in Wuchereria bancrofti infection

The elimination of microfilariae of Wuchereria bancrofti is probably mediated by free radicals. Red cell catalase (C), glutathione peroxidase (GPX), and superoxide dismutase (SOD) activity levels were measured as an indirect method of assessing blood oxidant status in 29 asymptomatic microfilaraemics, 29 "endemic normals", and 29 controls living in a non-endemic area. Changes in the activity of these enzymes were also compared over a one month period in 22 asymptomatic microfilaraemics randomised to receive either single dose or 14 day treatment with diethyl carbamazine citrate (DEC). Red cell GPX activity levels were significantly higher in "endemic normals" when compared to mf positive cases and non-endemic controls. An early and significant increase in GPX activity (on days 3, 7 and 14 compared to pretreatment levels, p<0.01) was observed after DEC in both treatment groups. Increases in the activity of catalase and SOD became significant only on days 14 and 30 respectively. The percentage reduction in microfilaraemia correlated significantly with the percentage increase in GPX activity levels (R(2)=0.58, p=0.6 x 10(-5)). Our results may suggest a role for GPX related oxidant species in the elimination of microfilariae.     

Case report: effects of diethylcarbamazine and thiabendazole combination against Mansonella perstans filariasis

Mansonella perstans filariasis is widely present in Africa and equatorial America and its pathogenicity has been recently reconsidered. Effective treatment is lacking and there is no consensus on optimal therapeutic approach. We present the results of a new combination treatment against M. perstans filariasis. Two cases of M. perstans filariasis were treated with the combination of diethylcarbamazine (DEC) and thiabendazole. The treatment was able to significantly reduce microfilaria burden in a case and to achieve complete clearance of blood microfilariae in another case.     

Brugia malayi: intravenous injection of microfilariae in ferrets as an experimental method for occult filariasis

Microfilaremia, immune responses, and pathology were compared in ferrets infected with 100 third-stage larvae of Brugia malayi (subperiodic strain) or injected intravenously with 10(6) microfilariae. Ferrets (Mustela putorius furo) inoculated with third-stage larvae typically became patent during the third month after infection, with a mean patency of 123 +/- 25 (SE) days. Ferrets injected intravenously with microfilariae exhibited a relatively constant microfilaremia for 3-4 weeks and usually cleared microfilariae before the fourth month. Ferrets that cleared microfilariae after intravenous injection of microfilariae or after infection with third-stage larvae failed to become patent or became amicrofilaremic within 3 weeks after a challenge intravenous injection of 10(6) microfilariae. Clearance of circulating microfilariae was associated with eosinophilia and serum antibody specific for the microfilarial sheath in ferrets injected with microfilariae and in most ferrets infected with third-stage larvae. Ferrets infected with third-stage larvae and necropsied after clearance of microfilariae had tissue inflammatory reactions to microfilariae characteristic of occult filariasis (tropical eosinophilia) in man; these ferrets exhibited immediate cutaneous hypersensitivity and circulating reaginic antibody to antigens of microfilariae. In ferrets necropsied following two intravenous injections of microfilariae, the majority of ferrets examined within 10 days after clearance of microfilariae had visible liver lesions to microfilariae identical to those of the ferrets infected with third-stage larvae; immediate cutaneous hypersensitivity and reaginic antibody were not consistently detected in ferrets injected with microfilariae. Sera from ferrets that had cleared circulating microfilariae were transferred passively into ferrets made microfilaremic by intravenous injection of microfilariae. Sera with microfilarial sheath-reactive IgG antibody titers (greater than or equal to 1:200) and microfilarial agglutination titers (greater than or equal to 1:40) rapidly cleared injected microfilariae (less than 24 hr); this serum also cleared or greatly reduced circulating microfilariae established by an infection with third-stage larvae; only the IgG-containing fraction of the sera was active in immune clearance. Sera that cleared microfilariae of B. malayi did not clear circulating microfilariae of Dirofilaria immitis or prevent recurrence of circulating microfilariae of B. malayi in ferrets infected with adult filariae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Combination of DEC plus aspirin induced mitochondrial mediated apoptosis in filarial parasite Setaria cervi

Diethylcarbamazine (DEC) is the main drug used against lymphatic filariasis but it is only microfilaricidal. Hence there is an urgent need for adulticidal drug. Aspirin is known nonsteroidal anti-inflammatory drugs which can inhibit prostaglandin H synthase and also induces apoptosis. Studies presented in this paper demonstrated that exposure of worms to the combination of DEC plus aspirin (DEC + A) at 100 μM concentration irreversibly paralyzed adult worms as well as microfilariae within 2 h. Some of the apoptosis markers viz; DNA fragmentation with accompanying ladder formation, upregulation of Bax expression and decrease in Bcl-2 have suggested that the parasite may be killed due to mitochondrial mediated apoptosis. The levels of several apoptosis regulating proteins and enzymes have also shown to be altered. DEC + A treated worms showed significant decrease in prostaglandin H synthase activity (PGHS) and increase in the level of nitric oxide (NO) and cysteine proteases while glutathione (GSH) and peroxidase level was found to be decreased. NO is known inducer of mitochondrial mediated apoptosis and acts by increasing the permeability of mitochondrial membrane through Bax and allowing cytochrome c to release in cytosol, inducing caspases leading to apoptosis. The DEC + A concentration used in this study is much lower than recommended dose so its intake is safe. Here we report for the first time that combination of DEC and aspirin is more effective and could be used as an adulticidal for control of human filarial infections.     

An ultrastructural study on the encapsulation of microfilariae of Brugia pahangi in the haemocoel of Anopheles quadrimaculatus

Chen C. C. and Laurence B. R. 1985. An ultrastructural study on the encapsulation of microfilariae of Brugia pahangi in the haemocoel of Anopheles quadrimaculatus. International Journal for Parasitology15: 421–428. The encapsulation of microfilariae of Brugia pahangi in the haemocoel of Anopheles quadrimaculatus was studied ultrastructurally. The microfilariae was first seen enclosed in an acellular electron dense capsule as early as 10 min after the engorgement of the mosquitoes from a cat parasitized by filariae. Two hours later, the mosquito plasmatocytes spread onto and around the humoral capsule. A completed capsule, which was seen at 24–48 h, was composed of an inner humoral layer and outer cellular layer. After 1 week, some electron dense haemocytes were seen attached to the outer surface of the cellular layer. These results suggested that the encapsulation of microfilariae in the haemocoel of mosquitoes combines both humoral and cellular reaction; humoral encapsulation occurs first and cellular encapsulation takes place later. The significance of combined reactions of humoral and cellular encapsulation in the mosquito-filarial system is discussed with reference to the encapsulation reaction of other insects.