Addition Of Heat-inactivated Serum Research Articles

Natural IgG antibodies in normal rabbit serum are involved in killing of the ompP2 mutant of Haemophilus parasuis SC096 strain via the classical complement pathway

Serum resistance in Haemophilus parasuis strain SC096 has been shown to be dependent on expression of the outer membrane protein P2 (OmpP2) and loss of the ompP2 gene results in significantly greater sensitivity to both porcine and rabbit sera. However, the mechanism of complement activation by the serum sensitive ΔompP2 strain is unknown. In this study, the classical complement pathway is demonstrated to be the main pathway for killing the H. parasuis ΔompP2 strain, and not the mannan-binding lectin (MBL) or alternative pathway. In addition, absorption of antibodies against ΔompP2 strain or depletion of IgGs from serum inhibited serum killing activity, which could be restored by addition of heat-inactivated serum or purified IgGs. Western blot analysis indicated that the OmpP2 mutant could bind significantly more IgGs than the wild type strain SC096 when incubated with serum. Finally, IgGs in normal rabbit serum targeted to the OMPs, but not lipooligosaccharide (LOS) in the OmpP2 mutant strain were found to be involved in bacterial killing indicating that the bactericidal epitope(s) is in the outer membrane proteins.

Testing of viscous anti-HIV microbicides using Lactobacillus

The development of topical microbicides for intravaginal use to prevent HIV infection requires that the drugs and formulated products be nontoxic to the endogenous vaginal Lactobacillus. In 30min exposure tests we found dapivirine, tenofovir and UC781 (reverse transcriptase inhibitor anti-HIV drugs) as pure drugs or formulated as film or gel products were not deleterious to Lactobacillus species; however, PSC-RANTES (a synthetic CCR5 antagonist) killed 2 strains of Lactobacillus jensenii. To demonstrate the toxicity of formulated products a new assay was developed for use with viscous and non-viscous samples that we have termed the Lactobacillus toxicity test. We found that the vortex mixing of vaginal Lactobacillus species can lead to reductions in bacterial viability. Lactobacillus can survive briefly, about 2s, but viability declines with increased vortex mixing. The addition of heat inactivated serum or bovine serum albumin, but not glycerol, prevented the decrease in bacterial viability. Bacillus atrophaeus spores also demonstrated loss of viability upon extended mixing. We observed that many of the excipients used in film formulation and the films themselves also afford protection from the killing during vortex mixing. This method is of relevance for toxicity for cidal activities of viscous products.

Effects of adsorbed heat labile serum proteins and fibrinogen on adhesion and apoptosis of monocytes/macrophages on biomaterials.

A previously established human monocyte culture protocol was used to determine the effects of varying adsorbed proteins on monocyte/macrophage adhesion and survival on dimethyl-silane (DM) or RGD modified glass coverslips. Cells were allowed to adhere for 2 h in the absence of protein or in the presence of serum, fibrinogen (Fg), heat inactivated serum (HIS), serum supplemented with Fg or HIS with Fg. Cell adhesion and apoptosis rates were determined on days 0 (2 h), 3, 7 and 10 of culture. The presence of serum alone in the initial culture was sufficient to optimize monocyte/macrophage adhesion and survival rates. Adding Fg to serum did not increase adhesion nor decrease apoptotic rates. No protein or the addition of HIS during the initial incubation period significantly decreased monocyte/macrophage adhesion and survival on both surfaces, however, the addition of Fg to HIS restored adhesion and survival rates to those seen with in the presence of serum alone on RGD surfaces. These studies demonstrate that monocyte/macrophage adhesion and survival on biomaterial surfaces are optimized by adsorbed heat labile serum proteins while adsorbed Fg plays a surface property-dependent role.

Demonstration of C3d and the terminal complement complex on thymic large granular lymphocytes and common thymocytes after incubation with naturally thymocytotoxic serum.

Guinea pig thymocytes were incubated in vitro with normal human or rabbit serum, which resulted in lysis of a major part of the cells, or with autologous serum causing lysis of a fraction (30%) of the cells. By using antibodies against human C3d and a neoepitope on the terminal complement complex (TCC), activation of both the initial and terminal part of the complement cascade was demonstrated on the surface of thymocytes incubated in the presence of serum. With human serum both types of antigen were detected. With rabbit serum only TCC was detected since immunoglobulins were bound to thymocytes and prevented specific demonstration of C3d by the antirabbit secondary antibody. With autologous serum only C3d was demonstrated, due to lack of cross-reactivity of the monoclonal anti-TCC antibody with guinea pig. Heat-inactivated sera or human serum devoid of IgM neither caused lysis nor resulted in complement activation. Addition of heat-inactivated serum restored the complement activating ability of IgM-depleted serum, indicating that heat stabile IgM is an obligate but sufficient requirement for complement activation in this system. The TCC epitope was also identified on a considerable number of granulated cells, on the basis of morphology classified as large granular lymphocytes.

Leishmania species: Mechanisms of complement activation by five strains of promastigotes

The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA Mg 2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This “naturally occurring” antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade, we are unable to visualize surface bound C3 on L. donovani promastigotes unless heat inactivated serum is also present. We conclude that all Leishmania spp. promastigotes are susceptible to lysis by normal serum independent of antibody. The presence of small amounts of naturally occurring antibody in human serum enhances the susceptibility of L. donovani promastigotes to lysis by activating the classical complement pathway.

Development of Microphalloides japonicus (Osborn, 1919) metacercariae in vitro (Trematoda: Microphallidae).

Metacercariae of the trematode Microphalloides japonicus (Osborn, 1919) were cultivated in various media to gravid adults, and the results were compared with those in vivo. A few eggs were produced in the uterus even in balanced salt solution, although cultivation in Eagle's MEM and NCTC 109 media was much better. Addition of heat-inactivated serum resulted in even better development. Encysted metacercariae also produced eggs in some media, but most of them were incomplete and without shells.