Drivers of hibernation in the brown bear.

BACKGROUND: Hibernation has been a key area of research for several decades, essentially in small mammals in the laboratory, yet we know very little about what triggers or ends it in the wild. Do climatic factors, an internal biological clock, or physiological processes dominate? Using state-of-the-art tracking and monitoring technology on fourteen free-ranging brown bears over three winters, we recorded movement, heart rate (HR), heart rate variability (HRV), body temperature (Tb), physical activity, ambient temperature (TA), and snow depth to identify the drivers of the start and end of hibernation. We used behavioral change point analyses to estimate the start and end of hibernation and convergent cross mapping to identify the causal interactions between the ecological and physiological variables over time. RESULTS: To our knowledge, we have built the first chronology of both ecological and physiological events from before the start to the end of hibernation in the field. Activity, HR, and Tb started to drop slowly several weeks before den entry. Bears entered the den when snow arrived and when ambient temperature reached 0 °C. HRV, taken as a proxy of sympathetic nervous system activity, dropped dramatically once the bear entered the den. This indirectly suggests that denning is tightly coupled to metabolic suppression. During arousal, the unexpected early rise in Tb (two months before den exit) was driven by TA, but was independent of HRV. The difference between Tb and TA decreased gradually suggesting that bears were not thermoconforming. HRV increased only three weeks before exit, indicating that late activation of the sympathetic nervous system likely finalized restoration of euthermic metabolism. Interestingly, it was not until TA reached the presumed lower critical temperature, likely indicating that the bears were seeking thermoneutrality, that they exited the den. CONCLUSIONS: We conclude that brown bear hibernation was initiated primarily by environmental cues, but terminated by physiological cues.

Antigen-activated T cells induce IL-12p75 production from dendritic cells in an IFN-γ-independent manner.

The addition of IL-12p75 to naïve CD4(+) T cells promotes their differentiation towards a TH1-type cytokine pattern. Dendritic cells stimulated by LPS generate IL-12p75, but only if the environment also contains IFN-γ. Thus, it appears that IFN-γ is needed to start the response that will result in further production of IFN-γ. We previously reported that paradoxically DCs produce IL-12p75 only after engaging primed, but not naïve T cells. This study examines the mechanism by which primed T cells trigger IL-12p75 secretion and asks whether this induction is also dependent on the presence of IFN-γ. Here, we show that, in contrast to LPS, primed T cells induce IL-12p75 in an IFN-γ-independent manner. Addition of rIFN-γ to cocultures of naïve T cells with DCs did not induce IL-12p75. Moreover, antigen-activated CD4(+) T cells from wild type or IFN-γ-deficient mice both initiated IL-12p75 production from DCs. Surprisingly, we found that synergies between three T-cell-derived factors - CD40 Ligand, IL-4 and GM-CSF - were necessary and sufficient for IL-12p75 production. These results suggest that there are at least two distinct pathways for IL-12p75 production in vivo. Furthermore, the T-cell-dependent pathway of IL-12p75 production employs molecules that are not classically associated with a TH1-type response.

A protective merozoite protein of Plasmodium falciparum shares an epitope with surface antigens of Paramecium.

A Plasmodium falciparum cDNA expression clone, lambdaPf9, had been identified earlier as a protective epitope, using anti-lambdaPf9 antibodies and combinatorial phagotopes. A segment of the Pf9 gene showed homology with Paramecium immobilization surface antigens such as 51B, 51A and 156G. A synthetic Pf9-peptide was designed from this region, and specific antibodies were raised. Each of these anti-Pf9 antibodies and combinatorial reagents, as well as anti-Paramecium 51B antibodies, recognized the Pf9-peptide on ELISA, and the same protein band in parasite immunoblots. The P. falciparum protein was released from the merozoite membrane fraction on treatment with PI-PLC, indicating the presence of a GPI anchor. Anti-Pf9-peptide antibodies specifically inhibited the growth of P. falciparum in culture. Immunofluorescence assays showed the reactivity of anti-Pf9-peptide sera with P. falciparum merozoites and gametocytes, as well as on the surface of Paramecium tetraurelia. The Pf9-peptide was able to induce proliferation of splenic lymphocytes obtained from mice infected with the rodent malarial parasites Plasmodium berghei and Plasmodium yoelii. These results point towards Plasmodium Pf9 as a conserved novel protective protein, sharing an epitope with Paramecium surface antigens.

Characterization of a differential immunoscreen epitope of Plasmodium falciparum using combinatorial agents.

A differential serological screening of a lambdagt11 cDNA expression library has identified several clones, which react exclusively to sera samples from persons clinically immune to malaria but not to acute malaria patient sera. One such clone, IPf9, has a 315-bp cDNA insert, which was found to be conserved in different strains of the human and rodent malarial parasite Plasmodium falciparum and Plasmodium berghei, respectively. The induced expression product of IPf9 was used to generate polyclonal sera in rabbits. The IPf9 expression product was also screened with phage surface display combinatorial libraries to isolate reagents that specifically bound to the IPf9 product. The polyclonal antisera and the combinatorial reagents recognized a 50-kDa protein from P. falciparum, and a 53-kDa product from P. berghei. Immunofluorescence studies using asexual and sexual stages of P. falciparum showed the protein to be present within the parasite in each of the asexual and sexual stages. The combinatorial reagents showed a partial inhibition in the growth of P. falciparum in vitro. Mice infected with the P. berghei showed the presence of T-cells that exhibited lymphoproliferation when stimulated with the IPf9 protein. It is suggested that IPf9 protein is a conserved protein epitope, and may be relevant for a protective immune response to malaria.

Antibodies against ribosomal phosphoprotein P0 of Plasmodium falciparum protect mice against challenge with Plasmodium yoelii.

Antibodies against the Plasmodium falciparum P0 ribosomal phosphoprotein (PfP0) have been detected exclusively but extensively in malaria-immune persons. Polyclonal rabbit and mice sera were raised against two recombinant polypeptides of P. falciparum P0 protein, PfP0N and PfP0C, covering amino acids 17 to 61 and the remaining amino acids 61 to 316, respectively. Sera against both these domains detected a 35-kDa protein from Plasmodium yoelii subsp. yoelii, a rodent malarial parasite, and stained the surface of merozoites in immunofluorescence assays. Total immunoglobulin G (IgG) purified from rabbit and mouse anti-PfP0 sera by ammonium sulfate and DEAE-cellulose chromatography was used for passive transfer experiments in mice. Mice passively immunized with both anti-PfP0N and anti-PfP0C showed distinctly lower levels of parasitemia than control mice. With immunizations on days -1, 0, 1, 3, and 5, about 50% of both sets of mice receiving anti-PfP0N and anti-PfP0C cleared the lethal 17XL strain of P. yoelii and revived by day 25. All the control mice died by day 10. By extending the immunization schedule, the survival period of the mice could be extended for every mouse that received anti-PfP0 IgG. These data demonstrate the cross-protection of the anti-PfP0 IgG and establish parasite P0 protein as a target for invasion-blocking antibodies.

Immunogenicity of the nonrepetitive regions of the circumsporozoite protein of Plasmodium knowlesi.

The circumsporozoite antigen (CS) of the simian malarial parasite Plasmodium knowlesi consists of tandemly repeated immunodominant peptide units that are variable and may play a role in evading the immune system. To study the immunogenicity of this antigen in the absence of the immunodominant repeats, the entire nonrepetitive region of the antigen was expressed in Escherichia coli as two fusion proteins with glutathione-S-transferase (GST) representing the amino terminal (GST-CSN) and the carboxy terminal domains (GST-CSC) of the CS antigen. The immunogenicity of these fusion proteins was studied in rabbits and different strains of mice. Antibody raised against both the CSN and CSC domains in both rabbits and every strain of mice recognized the native protein, as detected by immunofluorescence assay (IFA) using P. knowlesi sporozoites. A positive IFA reaction was also obtained with P. vivax sporozoites using antisera raised against the CSC domain. High titer antisera were raised in rabbits against both the domains, whereas mice showed comparatively low titers. On Western blots, mice showed specific response against the CSC domain. In both rabbits and mice, significant titers of antibodies were raised against region II, which has been shown to be the putative sporozoite binding site for hepatocytes in the case of P. falciparum.

Hydrous zirconium oxide as an anion-exchanger.

Samples of hydrous zirconium oxide have been prepared under varying conditions of precipitation, and their anion-exchange properties are reported. The effect of electrolyte concentration, time of equilibration and drying temperature on anion-exchange capacity has been studied. Stoichiometry of exchange, chemical stability and regeneration power of the exchanger have also been investigated. Distribution coefficients of various anions are reported, and some pairs of anions have been separated on the basis of the sorption data.