Host biomarkers and biological pathways that are associated with the expression of experimental cerebral malaria in mice.

Cerebral malaria (CM) is a primary cause of malaria-associated deaths among young African children. Yet no diagnostic tools are available that could be used to predict which of the children infected with Plasmodium falciparum malaria will progress to CM. We used the Plasmodium berghei ANKA murine model of experimental cerebral malaria (ECM) and high-density oligonucleotide microarray analyses to identify host molecules that are strongly associated with the clinical symptoms of ECM. Comparative expression analyses were performed with C57BL/6 mice, which have an ECM-susceptible phenotype, and with mice that have ECM-resistant phenotypes: CD8 knockout and perforin knockout mice on the C57BL/6 background and BALB/c mice. These analyses allowed the identification of more than 200 host molecules (a majority of which had not been identified previously) with altered expression patterns in the brain that are strongly associated with the manifestation of ECM. Among these host molecules, brain samples from mice with ECM expressed significantly higher levels of p21, metallothionein, and hemoglobin alpha1 proteins by Western blot analysis than mice unaffected by ECM, suggesting the possible utility of these molecules as prognostic biomarkers of CM in humans. We suggest that the higher expression of hemoglobin alpha1 in the brain may be associated with ECM and could be a source of excess heme, a molecule that is considered to trigger the pathogenesis of CM. Our studies greatly enhance the repertoire of host molecules for use as diagnostics and novel therapeutics in CM.

Non-opsonising aggregates of IgG and complement in haemoglobin C erythrocytes.

Haemoglobin C (HbC) differs from normal HbA by a lysine for glutamate substitution at position 6 of beta-globin. Heterozygous AC and homozygous CC phenotypes are associated with shortened erythrocyte life spans and mild anaemia. AC and CC erythrocytes contain elevated amounts of membrane-associated haemichromes, band 3 clusters, and immunoglobulin G (IgG) in vivo. These findings led us to investigate whether AC and CC erythrocytes might expose elevated levels of IgG and complement, two opsonins that have been implicated in the phagocytic clearance of senescent and sickle erythrocytes. Surprisingly, we found IgG, complement, and other plasma proteins co-localised in aggregates beneath the membrane of circulating AC and CC erythrocytes. These observations, and our finding of similar aggregates in erythrocytes heterozygous or homozygous for haemoglobin S (sickle-cell haemoglobin), suggest that the vast majority of membrane-associated IgG and complement detected in these abnormal erythrocytes is intracellular and does not contribute to the eventual opsonic clearance of these cells. Phagocytosis studies with macrophages provide evidence in support of this suggestion. Studies of erythrocyte clearance that involve the detection of membrane-associated IgG and complement as putative opsonins should investigate the possibility that these plasma proteins reside in the erythrocyte interior, and not on the cell surface.

Pathology of immunodeficient mice with naturally occurring murine norovirus infection.

Murine norovirus (MNV) was recently discovered in Rag2-/-/Stat1-/- mice in a U.S. medical research facility. Presently, little is known concerning the epidemiology and natural history of this virus. We studied the pathology of naturally occurring MNV infection in 28 immunodeficient mice of several different genotypes (Rag1-/-/IFNgamma R-/-, OT1 Rag1-/-/IFNgamma R-/-, OT2 Rag1-/-/IFNgamma R-/-, Rag1-/-/Stat1-/-, and Rag2-/-) that were maintained in two U.S. research facilities. The mice were selected for study because sentinel mice housed in their holding rooms had been identified as positive for MNV-specific antibodies during routine screening for infectious agents. Our data indicate that in certain lines of immunodeficient mice, MNV can establish a disseminated infection that is characteristically associated with inflammation in multiple tissues, including liver (hepatitis), lung (focal interstitial pneumonia) and the peritoneal and pleural cavities. In addition, MNV can establish an asymptomatic infection in the mesenteric lymph nodes of Rag2-/- mice. Further studies are needed to determine whether MNV presents a confounding variable in immunological, toxicological and pathological studies in mice naturally infected with MNV.

Immunohistochemical markers for the rodent immune system.

The responses to insults including chemical toxins, irradiation and infectious agents involve morphologic, biochemical and molecular changes in the immune system. The changes in specific tissues and cells often can be detected by histopathology and its associated field of immunohistochemistry (IHC). Cells normally express specific proteins (antigens) that can be detected by IHC. When responses to xenobiotics occur, cells often up or down regulate proteins. The art of IHC requires specialized procedures for detection of antigens. Fixation, tissue processing, immunoreactions and antigen retrieval methods are important elements of IHC. We review the antibodies, their sources, use of frozen or fixed paraffin-embedded tissues and specific IHC methods including antigen retrieval and illustrate how they can be effectively used to characterize the immunotoxicologic effects of agents.