Non-radioactive detection of K-ras mutations by nested allele specific PCR and oligonucleotide hybridization.

The development of methods to detect point mutations has been rapid over the recent years. In human colon tumours, a significant percentage of mutations are found in the K-ras gene. Faster and more sensitive methods for detection of these mutations are important for patient management and treatment. The author has extended the specificity of allele amplification of point mutations by using mismatch oligonucleotide primers in a polymerase chain reaction (PCR). It is shown that in colon tumours, the sensitivity of detecting a mutation is significantly higher when K-ras exon 1 is amplified prior to PCR with allele specific primers (mismatch PCR). It is shown that allele specific oligonucleotide probes which are non-radioactively labelled could be used to detect these point mutations. By utilizing this two-step PCR, K-ras codon 12 mutations were studied in 10 colon carcinoma cell lines and 25 colon tumours. By combining the two-step PCR together with non-radioactively labelled oligonucleotide probes, the detection of point mutations is both accurate and rapid.

Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes: role of bacterial membrane lipid.

Granule contents from human polymorphonuclear leukocytes were prepared by extraction with 0.2 M acetate, pH 4. A buffer extract fraction (peak D) obtained by Sephadex G-100 column chromatography demonstrated distinct antimicrobial activity toward Acinetobacte sp. independent of added H2O2 or Cl-. The protein of this fraction had an apparent molecular weight of 9,000 and demonstrated time and dose dependence that was more active against stationary-growth cells than mid-log-phase cells. The bactericidal activity of the fraction was most active at 37 degrees C, with only slight activity demonstrated at 22 degrees C and no activity at 4 degrees C. Boiling the granule fraction for 30 min did not affect the antimicrobial activity. However, pronase or trypsin pretreatment of the peak D fraction reduced its antimicrobial activity. When the membrane lipid composition of Acinetobacter sp. was altered by growth on specific n-alkane carbon sources, the susceptibility to the granule fraction was also altered. Resistance to the activity of the granule fraction increased as the carbon chain length of the growth substrate increased. Liposomes formed from Acinetobacter sp. lipid extracts and containing glucose were made leaky with the addition of the granule fraction (boiled and not boiled), suggesting a membrane-disruptive activity of the granule protein against Acinetobacter sp. membranes.

Effects of opsonization and gamma interferon on growth of Brucella melitensis 16M in mouse peritoneal macrophages in vitro.

Entry of opsonized pathogens into phagocytes may benefit or, paradoxically, harm the host. Opsonization may trigger antimicrobial mechanisms such as reactive oxygen or nitric oxide (NO) production but may also provide a safe haven for intracellular replication. Brucellae are natural intramacrophage pathogens of rodents, ruminants, dogs, marine mammals, and humans. We evaluated the role of opsonins in Brucella-macrophage interactions by challenging cultured murine peritoneal macrophages with Brucella melitensis 16M treated with complement- and/or antibody-rich serum. Mouse serum rich in antibody against Brucella lipopolysaccharide (LPS) (aLPS) and human complement-rich serum (HCS) each enhanced the macrophage uptake of brucellae. Combinations of suboptimal levels of aLPS (0. 01%) and HCS (2%) synergistically enhanced uptake. The intracellular fate of ingested bacteria was evaluated with an optimal concentration of gentamicin (2 microg/ml) to control extracellular growth but not kill intracellular bacteria. Bacteria opsonized with aLPS and/or HCS grew equally well inside macrophages in the absence of gamma interferon (IFN-gamma). Macrophage activation with IFN-gamma inhibited replication of both opsonized and nonopsonized brucellae but was less effective in inhibiting replication of nonopsonized bacteria. IFN-gamma treatment of macrophages with opsonized or nonopsonized bacteria enhanced NO production, which was blocked by N(G)-monomethyl L-arginine (MMLA), an NO synthesis inhibitor. MMLA also partially blocked IFN-gamma-mediated bacterial growth inhibition. These studies suggest that primary murine macrophages have limited ability to control infection with B. melitensis, even when activated by IFN-gamma in the presence of highly opsonic concentrations of antibody and complement. Additional cellular immune responses, e.g., those mediated by cytotoxic T cells, may play more important roles in the control of murine brucellosis.

Impaired control of Brucella melitensis infection in Rag1-deficient mice.

After intranasal inoculation, Brucella melitensis chronically infects the mononuclear phagocyte system in BALB/c mice, but it causes no apparent illness. Adaptive immunity, which can be transferred by either T cells or antibody from immune to naive animals, confers resistance to challenge infection. The role of innate, non-B-, non-T-cell-mediated immunity in control of murine brucellosis, however, is unknown. In the present study, we documented that BALB/c and C57BL/6 mice had a similar course of infection after intranasal administration of 16M, validating the usefulness of the model in the latter mouse strain. We then compared the course of infection in Rag1 knockout mice (C57BL/6 background) (referred to here as RAG-1 mice) which have no B or T cells as a consequence of deletion of Rag1 (recombination-activating gene 1), with infection in normal C57BL/6 animals after intranasal administration of B. melitensis 16M. C57BL/6 mice cleared brucellae from their lungs by 8 to 12 weeks and controlled infection in the liver and spleen at a low level. In contrast, RAG-1 mice failed to reduce the number of bacteria in any of these organs. From 1 to 4 weeks after inoculation, the number of splenic bacteria increased from 2 to 4.5 logs and remained at that level. In contrast to the consistently high numbers of brucellae observed in the spleens, the number of bacteria rose in the livers sampled for up to 20 weeks. Immunohistologic examination at 8 weeks after infection disclosed foci of persistent pneumonia and large amounts of Brucella antigen in macrophages in lung, liver, and spleen in RAG-1, but not C57BL/6, mice. These studies indicate that T- and B-cell-independent immunity can control Brucella infection at a high level in the murine spleen, but not in the liver. Immunity mediated by T and/or B cells is required for clearance of bacteria from spleen and lung and for control of bacterial replication in the liver.

Protection of mice against brucellosis by vaccination with Brucella melitensis WR201(16MDeltapurEK).

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of the conjunctiva or traumatized skin by infected animal products. A vaccine to protect humans from occupational exposure or from zoonotic infection in areas where the disease is endemic would reduce an important cause of morbidity worldwide. Vaccines currently used in animals are unsuitable for human use. We tested a live, attenuated, purine-auxotrophic mutant strain of Brucella melitensis, WR201, for its ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M. Mice inoculated intraperitoneally with WR201 made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 (IL-2), gamma interferon, and IL-10 when cultured with Brucella antigens. Immunization led to protection from disseminated infection but had only a slight effect on clearance of the challenge inoculum from the lungs. These studies suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis.