Arch Pathol Lab Med—Vol 123, February 1999 Human Albumin in W bancrofti—Peixoto et al 173 Immunocytochemical Localization and Distribution of Human Albumin in Wuchereria bancrofti Adult Worms Christina Alves Peixoto, MSc, PhD; Joaquim Noro ˜es, MD; Abraham Rocha, MSc; Gerusa Dreyer, MD ● To determine whether albumin is present on adult worms of Wuchereria bancrofti, thin sections of resin-em- bedded parasites were incubated with a specific antiserum to human albumin. With the exception of the epicuticle, all layers of the cuticle and the hypodermis were intensely labeled. Concentration of gold particles was observed within infoldings of the hypodermal membrane. Moderate labeling of the thin basement membrane that lines the pseudocelomic cavity and the gonoduct was also observed. Within the uterus, ovular membranes labeled intensely; groups of organized particles were seen below ovular membranes and also within invaginations of microfilarial embryos. In contrast, few gold particles were seen on the surface of mature intrauterine microfilariae. No labeling was observed in control sections incubated with antiserum preadsorbed with purified human albumin. The findings suggest that human albumin may be essential for the nu- trition and development of W bancrofti microfilariae. (Arch Pathol Lab Med. 1999;123:173–177) W uchereria bancrofti is the main parasite responsible for human lymphatic filariasis in tropical and subtrop- ical countries, causing approximately 120 million infec- tions worldwide. 1 The adult worms reside within human lymphatics, where they can survive for many years with- out causing clinical disease. Thus, the parasites are well adapted to survive within the mammalian host, but the nature of the mechanisms that permit adult worms to evade protective host responses and reproduce within the lymphatic system are largely unknown. Development of immune tolerance to filarial worms has been invoked to explain the persistence of microfilaremia. However, it is unclear whether adult worms themselves induce immune unresponsiveness, because adult worm carriers without microfilaremia tend to have vigorous antifilarial immune responses. 2,3 The masking of surface antigens by host-derived mol- ecules is thought to be one mechanism of immune evasion by schistosomes. 4 Human serum albumin may play a sim- Accepted for publication August 21, 1998. From the Departments of Pathology and Cellular Biology (Dr Peixoto) and Parasitology (Drs Peixoto and Dreyer and Mr Rocha), Centro de Pesquisas Aggeu Magalha ˜es, and the Department of Surgery, Section of Urology, Centro de Ciencias da Saude (Dr Noro ˜ es), Universidade Federal de Pernambuco, Recife, Brazil. Reprints: Christina Alves Peixoto, MSc, PhD, Department of Pathol- ogy and Cellular Biology, Centro de Pesquisas Aggeu Magalha ˜es, Av Moraes Rego s/n, CEP 50670-420 Recife, Brazil. ilar role in bancroftian filariasis, because it is a major com- ponent of the surface of W bancrofti microfilariae. 5 In the present study, we examined the distribution of human al- bumin in adult worms of W bancrofti surgically removed from the intrascrotal lymphatics of a volunteer. MATERIAL AND METHODS Parasites Living adult W bancrofti were obtained from a volunteer par- ticipant in a study of the adulticidal effect of diethylcarbamazine. Preoperative ultrasonography 6 had shown the presence of an in- trascrotal lymphatic dilation of 20 mm containing living adult worms, which remained alive after 12 days of treatment with diethylcarbamazine (6 mg/kg per day). The patient consented to surgery for the collection of lymph fluid to measure local dieth- ylcarbamazine levels and for the removal of adult worms. The study protocol and procedures were reviewed and approved by the Ethical Committee of the Hospital das Clinicas, Universidade Federal de Pernambuco, Recife, Brazil. Electron Microscopy and Immunocytochemistry The living parasites were washed twice with phosphate-buff- ered saline (PBS) and fixed for 60 minutes in a solution contain- ing 0.1% glutaraldehyde and 4% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 mol/L cacodylate buffer, pH 7.2. After fixation, the parasites were washed twice with PBS, dehy- drated in acetone, and embedded in L. R. White resin (Sigma Chemical Company, St Louis, Mo). Because of its hydrophilic na- ture, this resin is highly suitable for labeling thin sections with colloidal gold probes. Polymerization was done at 37°C for 5 days. Ultrathin sections were collected on 300-mesh nickel grids and incubated for 60 minutes at room temperature in PBS, pH 8.0, con- taining 5% nonfat milk and 0.01% Tween 20 (PBS-MT). The sections were then incubated for 1 hour at room temperature with rabbit immunoglobulin G (IgG) anti-human albumin diluted 1:500 in PBS- MT. The sections were then rinsed in PBS and incubated for 1hour at room temperature with gold-labeled goat anti-rabbit IgG (Sigma) diluted 1:100 with PBS-MT. The specificity of the antibody against human albumin was ascertained by incubation for 2 hours of aso- lution containing 2% purified human albumin in the presence of rabbit IgG anti-human albumin (Sigma) at the final dilution of 1: 500 in PBS, pH 8.0. The adsorbed antibody was pelletized at 12000g for 30 minutes, and the supernatant was used for immunocytochem- ical analysis. Second antibody control sections were incubated only in the presence of the gold-labeled antibody. After incubation, the sections were washed with PBS followed by distilled water, coun- terstained with uranyl acetate and lead citrate, and examined with a transmission electron microscope (Zeiss Em 109, Schott-Zeiss do Brasil LTDA, Rio de Janeiro). RESULTS AND COMMENT In this study, human albumin was readily demonstrated in all layers of the cuticle and in the hypodermis of adult