Body Temperature files

Text files containing the body temperature recordings from individual female laboratory mice housed singly, in pairs, or in groups of 5 in laboratory cages. Temperature is in Celsius (C = Celsius) and was recorded using iButton temperature dataloggers. Each line contains the date and time of the body temperature recording. The first number in the filename represents the mouse ID for that file. Singlets: SC4.1, SC10.2, SC17.4, SC35.8, SC41.10, SC43.10, SC54.12, SC56.13, SC61.14. Pairs: (SC2.1 with SC19.5), (SC7.2 with SC16.4), (Sc5.1 with SC22.5), (SC27.1 with SC60.14), (SC33.8 with SC45.11), (SC39.9 with SC59.13), (SC44.10 with SC50.12), (SC30.7 with SC46.11). Quintets: (SC3.1, SC6.2, SC13.3, SC15.4, SC23.5), (SC8.2, SC11.3, SC14.4, SC21.5, SC25.6), (SC1.1, SC9.2, SC18.4, SC20.5, SC24.6), (SC26.7, SC38.9, SC42.10, SC49.11, SC52.12), (SC31.8, SC40.9, SC47.11, SC51.12, SC58.13), (SC34.8, SC36.9, SC53.12, SC55.13, SC62.14), (SC29.7, SC32.8, SC37.9, SC48.11, SC57.13

Immune responses and MTB bacterial counts in mice immunised with BCG and boosted with ICM.

<p>Mice were immunised s.c. with BCG and twice boosted with ICM six and eight weeks later; 2 weeks after the final boost, mice were either culled and their tissues (blood and spleens) used for immunological evaluation, or challenged i.n. with 70,000 H37Rv. * Indicates statistically significant difference (p<0.05). A,B) Ag85B (A) and Acr (B) specific IgG, IgG1 and IgG2a serum responses determined by ELISA; shown are the mean values from 3 mice analysed in triplicates and in serial dilutions (indicated by differing patterns). C) Splenocyte proliferation after <i>in vitro</i> stimulation with Ag85B, measured by ³[H]-thymidine incorporation and expressed as stimulation indices (specific/nonspecific proliferation); n = 3 animals. D) IFN-γ release in splenocyte cultures (as in C) measured by an IFN-γ ELISA based kit. E) Lung bacterial counts in immunised mice; shown are the counts for individual mice (n = 6) and the means +/− SEM for each group.</p

Immune responses and MTB bacterial counts in mice immunised with ICM.

<p>Mice were immunised with 50 µg ICM (1∶20 antibody antigen ratio) or with Ag85B alone (30 µg), Ag85B+CT, BCG and PBS; two weeks after the final immunisation mice were either culled and their tissues (blood and spleens) used for immunological evaluation, or challenged i.n. with 70,000 MTB H37Rv. * Indicates statistically significant difference (p<0.05). A,B) Ag85B (A) and Acr (B) specific IgG, IgG1 and IgG2a serum responses determined by ELISA; shown are the mean values from 3 mice analysed in triplicates and in serial dilutions (indicated by differing patterns). C) Splenocyte proliferation after <i>in vitro</i> stimulation with Ag85B, measured by ³[H]-thymidine incorporation and expressed as stimulation indices (specific/nonspecific proliferation); n = 3 animals. D) IFN-γ release in splenocyte cultures (as in C) measured by an IFN-γ ELISA based kit. E) Lung bacterial counts in immunised mice; shown are the counts for individual mice (n = 6, except in some groups due to death of animals before the end of the experiment) and the means +/− SEM for each group.</p

Functional evaluation of ICM in vitro and in vivo.

<p>A) Complement C1q binding ELISA; ICM were used at the 1∶20 antibody-antigen ratio and the neat sample contained 5 µg/m total protein (ICM) or the equivalent amount for individual components; each bar represents mean value from triplicate assays and the patterns indicate serial dilutions. B) Analysis of binding of ICM to spleen-derived APCs by flow cytometry; shown are the proportions of cells (out of 10,000 counted) that bound either mAb alone or ICM. C) Serum anti-Ag85B IgG responses from mice immunised with an equimolar (1∶1) or a low (1∶20) antibody-antigen ratio, twice at the base of the tail, at 3-week intervals. Mice were culled 3 weeks after the final immunisation. Shown are the mean values and corresponding serial dilutions from a pilot experiment (n = 3 mice).</p

Expression, purification and chemical crosslinking of recombinant proteins.

<p>A) Coomassie Blue staining of purified, His-tagged proteins separated by 12% SDS-PAGE; 1. Acr-Ag85B (50 kDa), 2. Acr (20 kDa) and 3. Ag85B (32 kDa). B) Western blot analysis using antigen-specific antibodies; 1. Acr, 2. Acr-Ag85B and 3, Ag85B (1 and 2 probed with anti-Acr mAb TBG65; 3 probed with rabbit anti-Ag85B serum). C) Chemical crosslinking of Acr; shown is a Coomassie-stained (1, and 2) or Western blot (3 and 4) analysed sample with crosslinker (1 and 3) or without crosslinker (2 and 4). Letters indicate various molecular forms based on expected size (M-monomer, D-dimer, Tr-trimer, Te-tetramer, H-hexamer, O-oligomers). D) Chemical crosslinking of Acr-Ag85B fusion protein; shown is a sample with (1 and 3) or without (2 and 4) crosslinker. Letters indicate various molecular forms as for Acr (C). E) Chemical crosslinking of Ag85B (internal control); Coomassie and Western blot analysis of a sample with (1 and 3) or without (2 and 4) crosslinker.</p

Schematic representation of immune complex mimics (ICM) based on Acr-Ag85B fusion protein and an anti-Acr mAb (A) and the classical immune complexes (IC) based on Ag85B antigen of MTB and polyclonal Abs (B).

<p>For ICM, the fusion protein is depicted as a trimer, which is one of the predominant molecular forms for Acr in solution. Each mAb molecule must bind to a different monomer unit of Acr (A); in contrast, polyclonal Abs can bind to the same Ag85B molecule (B).</p