Cloning, large-scale production and characterization of fusion protein (P-TUFT-ALT-2) of Brugian abundant larval transcript-2 with tuftsin in Pichia pastoris.

Lymphatic filariasis is a "disease of poor people" due to a large section of affected people with economic backwardness. Therefore, successful elimination of this disease requires a cost-effective prophylactic agent such as vaccine along with conventional drugs. The Abundant Larval Transcript-2 (BmALT-2) protein of Brugia malayi has been recognized as the most potential vaccine candidate. Tuftsin, a tetra-peptide immunopotentiator has already shown the enhanced immunogenicity of various vaccine antigens in earlier studies. This study deals with the development of tuft-alt-2 fusion construct and a suitable culture condition for its large-scale production in Pichia pastoris. The recombinant P. pastoris/tuft-alt-2 with 9-11 copies of the gene construct exhibited the highest expression level. The molecular weight of P-TUFT-ALT-2 was determined as 28 kDa in SDS-PAGE including 3 kDa due to glycosylation. The dry cell biomass was 57.4 gL-1 in the bioreactor. The P-TUFT-ALT-2 expression was measured as about 35 mg L-1, which was 102% higher than flask culture. The P-TUFT-ALT-2 produced the highest 65,000 IgG peak titer in Balb/c mice. Moreover, P-TUFT-ALT-2 exhibited about 9.46% higher splenocyte proliferation than E. coli expressed E-ALT-2 alone. The enhanced secreted production of P-TUFT-ALT-2 in bioreactor would step up its commercialization as an inexpensive commercial vaccine for human lymphatic filariasis.

Autotransplant of spleen tissue in children with schistosomiasis: evaluation of splenic function after splenosis.

Autotransplantation of spleen tissue has been done, in the past ten years, in children with schistosomiasis mansoni with bleeding varices. The purposes of this investigation were: (1) to study the morphology and function of the remnant spleen tissue; (2) to quantify the production of tuftsin; and (3) to assess the immune response to pneumococcal vaccine of these patients. Twenty three children, who underwent splenectomy and autologous implantation of spleen tissue into the greater omentum were included in this investigation. The average postoperative follow-up is five years. Splenosis was proved by colloid liver-spleen scans. Search for Howell-Jolly bodies assessed the filtration function. Tuftsin and the titer of pneumococcal antibodies were quantified by ELISA. Splenosis was evident in all children; however, it was insufficient in two. Howell-Jolly bodies were found only in these two patients. The mean tuftsin serum concentration (335.0 +/- 29.8 ng/ml) was inside the normal range. The immune response to pneumococcal vaccination was adequate in 15 patients; intermediate in four; and inadequate in four. From the results the following conclusions can be drawn: splenosis was efficient in maintaining the filtration splenic function in more than 90% and produced tuftsin inside the range of normality. It also provided the immunologic splenic response to pneumococcal vaccination in 65% of the patients of this series.

Autotransplant of spleen tissue in children with schistosomiasis: evaluation of splenic function after splenosis

Autotransplantation of spleen tissue has been done, in the past ten years, in children with schistosomiasis mansoni with bleeding varices. The purposes of this investigation were: (1) to study the morphology and function of the remnant spleen tissue; (2) to quantify the production of tuftsin; and (3) to assess the immune response to pneomococcal vaccine of these patients. Twenty three children, who underwent splenectomy and autologous implantation of spleen tissue into the greater omentum were included in this investigation. The average postoperative follow-up is five years. Splenosis was proved by colloid liver-spleen scans. Search for Howell-Jolly bodies assessed the filtration function. Tuftsin and the titer of pneumococcal antibodies were quantified by ELISA. Splenosis was evident in all children; however, it was insufficient in two. Howell-Jolly bodies were found only in these two patients. The mean tuftsin serum concentration (335.0 ± 29.8 ng/ml) was inside the normal range. The immune response to pneumococcal vaccination was adequate in 15 patients; intermediate in four; and inadequate in four. From the results the following conclusions can be drawn: splenosis was efficient in maintaining the filtration splenic function in more than 90 percent and produced tuftsin inside the range of normality. It also provided the immunologic splenic response to pneumococcal vaccination in 65 percent of the patients of this series

The characteristics of purified HL60 tuftsin receptors.

The purification and characteristics of purified HL60 tuftsin receptors are described. Purification was accomplished by affinity chromatography similar to that described earlier, wherein a tuftsin analog Thr-Lys-Pro-Pro-Arg, is covalently linked at the N alpha group to a solid support. The receptor consists presumably of two subunits approximately 66 KDa and 57 KDa. The dissociation constant of the receptor complex is 4.7 X 10(-8) M with 5 X 10(4) receptors per cell. It can form oligomers with an Mr of about 560 KDa suggesting an octomeric structure, assuming the same number of each subunit is associated.

Effects of protizinic acid on leukokinin generation and its physiological action.

The effect of protizinic acid (PRT), a non-steroidal antiinflammatory drug, on the in vivo leukokinin (LK) generation system using feline acute ischemia model, in vitro LK generation system and the LK-induced contraction of the isolated smooth muscle was investigated. When 3 mg/kg PRT was injected twice intravenously to cats with acute cardiac ischemia, increased blood acid protease activity was inhibited and significant inhibitory action on the decrease of leukokininogen, the precursor of LK, was observed. Simultaneously, ST-segment elevation on the electrocardiogram tended to be suppressed and the lowered mean aortic blood pressure was significantly restored. On the LK generation induced by rabbit kininogen and acid protease derived from mouse L-1210 leukemic cells or rabbit polymorphonuclear leukocytes, PRT showed a dose-dependent inhibition while indomethacin (IM) and ibuprofen (IB) at a concentration of 3 X 10(-4) M showed no effect. However, potencies of the inhibitory actions of PRT, IM and IB on the LK generation induced by bovine spleen cathepsin D were almost the same at a concentration of 3 X 10(-4) M. Furthermore, PRT as well as IM showed antagonistic action on the isolated rat uterine contraction induced by LK. These results suggest that PRT not only inhibits the in vitro and in vivo generation of LK but also antagonizes to it on the receptor site of LK.

[Tuftsin]. / Tuftsin.

Literature review. The tetrapeptide tuftsin (Thr-Lys-Pro-Arg) is cleaved off the carrier IgG molecule enzymatically and stimulates the phagocytic and bactericidal activity of polymorphonuclear leucocytes and macrophages. The action of tuftsin is mediated by specific receptor sites on the surface of these cells. Its antitumor activity has been shown in vitro as well as in vivo. The influence of tuftsin on the major metabolic processes of the cell (hexosemonophosphate shunt; cAMP/cGMP; Ca++-distribution; redox reactions) is the basis of its mode of action.--The feature of tuftsin and its low toxicity make it a useful agent for immunotherapy.

Colonic surgery. The splenic connection.

Colonic resections, particularly those that require mobilization of the splenic flexure, occasionally will lead to injury of the spleen. Under these circumstances, the abdominal surgeon has traditionally considered incidental splenectomy to be the only safe alternative. Currently, a better understanding of splenic physiology and its role in sepsis prevention has reversed this trend. These efforts to preserve splenic function have resulted in various options available to the surgeon, herein reviewed. The results obtained in 36 general surgical patients with splenic injuries suggest that the salvage of the spleen is a safe alternative. In situations where salvage is impossible, the surgeon can resort to omental autotransplantation of the removed spleen, a recently described technique of appealing simplicity. The results obtained with this procedure in 23 other patients are presented.

Defective phagocytosis due to deficiencies involving the tetrapeptide tuftsin.

A description of the symptoms and causes of tuftsin deficiency is presented. Two major causes which give rise to a deficiency of the tetrapeptide (thr-lys-pro-arg) are discussed. One is familial tuftsin deficiency syndrome that results from an inherited mutation involving the tetrapeptide. All patients give a history of repeated infection with variable severity. One parent, father or mother, is deficient and occasionally other siblings may have the disease. The other type of deficiency is the result of loss of splenic function whether it is due to surgical removal of the spleen and to infarction or infiltration of the organ. A method for the assay of tuftsin activity is described.