Development of anti-bovine TNF-alpha mAb and ELISA for quantitating TNF-alpha in milk after intramammary injection of endotoxin.

Murine mAb reactive with recombinant bovine tumor necrosis factor-alpha (r-boTNF-alpha) were produced. An ELISA using murine mAb and rabbit polyclonal antibodies, each reactive with r-boTNF-alpha to sandwich bovine TNF-alpha was developed. Secretion of TNF-alpha in quarter milk increased 1 h after injection of 0.1 mg (four cows) or 0.5 mg (four cows) Escherichia coli lipopolysaccharide (LPS) into a mammary quarter, peaked 1 to 5 h later, and returned to control levels in 24 h. There were no differences in body temperature, SCC, TNF-alpha, and blood leukocyte responses between 0.1 and 0.5 mg of LPS. To determine effects of repeated injections of LPS into the same udder, a second injection of 0.1 mg of LPS into the same quarter (two cows) 24 h after the first injection produced a strongly attenuated TNF-alpha response. However, a normal TNF-alpha response was observed when LPS was injected into a contralateral quarter (two cows) 24 h after the first LPS injection. Leukocyte counts in blood decreased and body temperature increased substantially after each injection of LPS. Quarter milk SCC increased 200-fold 8 to 12 h after the LPS injections. It would appear that these changes were not regulated by TNF-alpha secretion because the changes were also similar after the second injection of LPS into the same mammary quarter.

A large toxin from pathogenic Escherichia coli strains that inhibits lymphocyte activation.

The mechanisms by which bacteria resist cell-mediated immune responses to cause chronic infections are largely unknown. We report the identification of a large gene present in enteropathogenic strains of Escherichia coli (EPEC) that encodes a toxin that specifically inhibits lymphocyte proliferation and interleukin-2 (IL-2), IL-4, and gamma interferon production in response to a variety of stimuli. Lymphostatin, the product of this gene, is predicted to be 366 kDa and shares significant homology with the catalytic domains of the large clostridial cytotoxins. A mutant EPEC strain that has a disruption in this gene lacks the ability to inhibit lymphokine production and lymphocyte proliferation. Enterohemorrhagic E. coli strains of serotype O157:H7 possess a similar gene located on a large plasmid. Loss of the plasmid is associated with loss of the ability to inhibit IL-2 expression while transfer of the plasmid to a nonpathogenic strain of E. coli is associated with gain of this activity. Among 89 strains of E. coli and related bacteria tested, lifA sequences were detected exclusively in strains capable of attaching and effacing activity. Lymphostatin represents a new class of large bacterial toxins that blocks lymphocyte activation.

Inhibition of murine splenic and mucosal lymphocyte function by enteric bacterial products.

Previously we showed that lysates of enteropathogenic Escherichia coli (EPEC) inhibit lymphokine production by mitogen-activated human peripheral blood and lamina propria mononuclear cells. The aims of the present study were to determine whether EPEC-inhibitory factors have similar effects on murine lymphoid populations in order to further delineate the mechanisms of alteration of cytokine production. Preexposure to EPEC lysates inhibited mitogen-stimulated interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) production by murine spleen cells, but IL-10 production was increased. The inhibition was not due to increased apoptosis and was not blocked by neutralizating antibodies against IL-10 or transforming growth factor beta (TGF-beta). EPEC lysates also inhibited mitogen-stimulated IL-2 and IFN-gamma production by CD11b-depleted spleen cells, IL-2 and IL-4 production by intraepithelial and Peyer's patch lymphocytes, IL-2 production by the human T-cell line Jurkat, and antigen-stimulated IL-2 production by murine spleen cells. Lysates obtained from Shiga-like toxin-producing E. coli, E. coli RDEC-1, Citrobacter rodentium, and an EPEC espB insertion mutant all inhibited IL-2 and IL-4 production by mitogen-stimulated lymphoid cells. In conclusion, lysates of EPEC and related bacteria directly inhibit cytokine production by lymphoid cells from multiple sites by a mechanism that does not increase apoptosis or result from secondary effects of IL-10 or TGF-beta.

Interaction of H-2Eb with an IAP retrotransposon in the A20/2J B cell lymphoma.

In the A20/2J BALB/c B cell lymphoma, Southern analysis revealed an insertion of approximately 6 kilobases of DNA into the first intron of one Ebd-allele. Two observations suggest that the rearrangement did not occur recently in the A20/2J subline. Firstly, normal and altered Ebd-alleles are present in equal numbers, and secondly, the LB 27.4 and LS 102.9 somatic cell hybrids formed at an earlier date both possess the rearrangement. Sequences of two cDNA clones, lambda Eb-7 and lambda Eb-125, selected from an A20/2J cDNA library prepared from poly [A+] RNA indicate that the rearranged Ebd-allele directs the synthesis of atypical Eb transcripts. The clones contain Eb sequence linked to a portion of retroviral-like intracisternal A-particle (IAP) genomic sequence, and they appear to be copies of mRNA produced by splicing between a 5' donor site in the retroviral transcript and the 3' acceptor site of the Eb gene's first intron. The longer lambda Eb-125 insert corresponds to RNA that initiated in the 5'-untranslated region of the Eb gene. The 3'-end of the first Eb exon joins to long terminal repeat sequence, and retroviral sequence extends up to the splice junction with the second Eb exon; 3' of the junction, the lambda Eb-125 sequence corresponds to that of a correctly spliced Eb transcript. It seems feasible that the cDNA clones represent hybrid RNA synthesized by read-through transcription of the Eb coding region and an IAP element inserted into the first intron of the rearranged Ebd-allele.

Production and characterization of monoclonal antibodies to porcine immunoglobulin gamma, alpha, and light chains.

Monoclonal antibodies (MAB) to porcine immunoglobulins were produced by fusion of SP2/0 cells with splenic lymphocytes of mice that had been immunized with porcine IgG or IgA. Of 16 MAB selected for detailed study, 13 reacted with heavy chains (7 anti-gamma, 6 anti-alpha) and 3 reacted with light chains of native immunoglobulin. Several of the same MAB (3 anti-gamma chain, 1 anti-alpha chain, 3 anti-light chain) also reacted with denatured immunoglobulin by use of immunoblotting analysis. Collective results of competitive ELISA, immunodiffusion, and immunoblotting analysis indicated that the 7 MAB of anti-gamma chain specificity were directed to 3 epitopes, the 6 MAB of anti-alpha chain specificity were directed to at least 2 epitopes, and the 3 MAB of anti-light chain specificity were directed to at least 2 epitopes that may have been located of different types of light chains. When tested by immunodiffusion with 5% polyethylene glycol incorporated in the agar matrix, all anti-gamma chain and antilight chain MAB, but no anti-alpha chain MAB, had precipitating activity. When polyethylene glycol was not used, only 4 MAB (all of the anti-gamma chain specificity and of IgM isotype) had precipitating activity. These MAB, with specificity for gamma and alpha chains and those reported earlier with mu-chain specificity, should be invaluable in the detection and quantification of porcine immunoglobulin isotypes. These MAB have potential applications in delineating the porcine immune response to selected immunogens.

Intramedullary fixation of implants pre-coated with bone cement: a preliminary study.

A preliminary study on the feasibility of implants pre-coated with an acrylic bone cement has been performed. Four types of implants, an actual canine femoral prosthesis, a polished steel rod (0.49 cm dia. x 13 cm long) with and without pre-coating, and a sand-blasted steel rod with pre-coating were implanted into canine femurs in vitro to evaluate the interfacial shear strengths. After serial sectioning the samples in discs, push-out tests were made. The weakest interfacial shear strength was exhibited by the polished rod/cement interface (0.5 MPa) while the strongest was the "old" and "new" cement interface (23.4 MPa). The bone/cement interfacial strength was in between (1.17 MPa). The shear strength of rod/cement interface increased substantially by sand-blasting (6.84 MPa or 585% increase). The proposed method increases the modified implant's interfacial shear strength by 337% (from 1.17 to 3.84 MPa) over the conventional implants. It may furthermore reduce the setting temperature, the shrinkage, and the amount of monomer released during operation due to the reduced amount of cement at the time of implantation. The more gradual transmission of load from implant to bone and "auto-centering" of implants during operation by pre-coating, are thought to be advantageous over conventional cement fixation method.