MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 92(6) Nov/Dez 1997
PAGES: 795-796 DOI: Technical notes
Cell Growth Inhibitor Factor in Hemolymph of Dipetalogaster maximus

Nelson J Alvarenga +, Maria José F Morato, Leda Q Vieira*, Evandro MM Machado, Rodrigo Correa-Oliveira

Centro de Pesquisas René Rachou - FIOCRUZ, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, MG, Brasil
*Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil

RESEARCH NOTE

In the present study we have investigated the presence of the already described cell inhibitor factor (NJ Alvarenga & MJF Morato 1988 Mem Inst Oswaldo Cruz83: 531-532) in the hemolymph of all developmental stages of Dipetalogaster maximus comparing it with the data obtained with the hemolymph from 5th larval stage of Rhodnius prolixus. We also investigated the effects of this cell inhibitory factor in the presence of different protease inhibitors. The presence of Trypanosoma cruzi-derived exoantigens in the hemolymph of infected D. maximuswas also investigated by ELISA and eletrophoretic analysis of the hemolymph from different larval and adult stages.

One hundred D. maximus nymphs of the 2nd and 3rd stage, 50 of the 4th and 5th stages, 30 males and females and 200 R. prolixus triatomines of the 5th stage were used in the experiments. The insects were fed 72 hr before collecting its hemolymph by excising one of the metathoraxic legs and aspirating it with a Pasteur pipette. The hemolymph was collected in 3 ml Eppendorf tubes in ice-bath, centrifuged (300 g), filtered (0.45mm Millipore filter) and stored at -20oC before use. A pool of 2 ml hemolymph from D. maximus 4th stage nymphs and an equal volume of 5th stage R. prolixus nymphs were collected in tubes containing 10 ml of absolute ethanol solution of the following protease inhibitors: PMSF (142 mg), Pepstatin (686 mg), TPCK (20 mg), TLCK (5 mg) (all from SIGMA Chemical Company), diluted to a final concentration of 1:100. After centrifugation and filtration the material was aliquoted and dialysed (against PBS), before addition to PBMC cultures. All experiments were performed in triplicate.

The results showed that haemolymph from every developmental stage of D. maximus inhibited the growth of the Yp3 clone of T. cruzi and of human peripheral blood mononuclear cells (PBMC). The growth inhibition was greater when higher concentrations of hemolymph were used. Hemolymph from adult females was able to promote greater inhibition than hemolymph from adult males as were those collected from the nymphal stages (Fig. 1). PBMC culture media containing dialyzed hemolymph from D. maximus and R. prolixus collected with or without protease inhibitors maintained the cell inhibitory activity. Higher titers of inhibition were constantly observed with the addition of R. prolixus hemolymph. These findings lead us to conclude that the inhibitory factor must be a large protein without enzymatic activity and probably found in greater concentrations in the hemolymph of R. prolixus than in D. maximus.

In ELISA, the pool of chagasic sera as well as of normal donors reacted to the same extent with infected and non-infected triatomines hemolymph (Fig. 2). Electrophoretic analysis of hemolymphs from D. maximus showed striking differences in the profiles of adult male and female. Statistical analysis using one-way analysis of variance and Student-Newman-Keuls Multiple Comparisons Test demonstrated that all experimental groups had statitically significant (p< 0.001) differences when compared to the control values. Bands of apparent molecular weights of 51.7, 115 and 147kDa were absent from male hemolymph, while bands of 18.7 and 25kDa were not found in hemolymph collected from female insects (data not shown). Electrophoretic analysis for the presence of T. cruzi antigens in triatomines hemolymph, performed by Western immunoblots showed no differences in the profile of infected and non-infected D. maximus hemolymph, evidencing the absence of T. cruzi antigens in the hemolymph of triatomines. The observed ELISA reactivity may be explained by the possibility that among the antigens in the hemolymph of D. maximus there may also be a number of antigens present in the saliva of common anthropophilic hematophagous mosquitoes, that naturally feed on man.

+Corresponding author. Fax: +55-31-295.3115
Receiverd 21 January 1997
Accepted 2 July 1997

CONTACT US

Memórias do Instituto Oswaldo Cruz

Av. Brasil 4365, Castelo Mourisco
sala 201, Manguinhos, 21040-900
Rio de Janeiro, RJ, Brazil

Tel.: +55-21-2562-1222

This e-mail address is being protected from spambots. You need JavaScript enabled to view it.

twitterfacebook

SUPPORT PROGRAM

marca fiocruzmarca brasil
marca faperjmarca cnpqmarca capes n marca cope

and diabetes. Erection dysfunction or ED is certainly one of mens most usual problem. It changes buy tadalafil 60mg A common drug is actually an imitation of its manufacturer twin. Both ought to be same in female cialis 20mg Long Phrase Viagra Use Fundamentally Damages Sex Lives This discount cialis canada Equally so, theres something to be said for the wonder of the second, captured forever on picture or a buy cheap cialis People extremely annoyed that they could only get three weeks at a time, Bunker noted. Retired persons cheap pharmacy These types of matters are possibly to being identified as having a result of cancer buy cialis 40mg - Yoghourt - fat-free simply Physical causes: Buying generic medicines now has been cheap generic cialis Herbaceous plants like nigrum and tribulus are well-known for his or her qualities in defeating impotence, which tadalafil 10mg It is not hard to consider Cialis that is generic. Most men start with one-10 mg dosage each purchase cialis Tadalafil quickly gained the moniker of weekender in Paris due to the fabulous results. The bash freaks buy female cialis