A Cholecystokinin Analogue Ameliorates Cognitive Deficits and Regulates Mitochondrial Dynamics via the AMPK/Drp1 Pathway in APP/PS1 Mice.

BACKGROUND: There are no drugs on the market that can reverse or slow Alzheimer's disease (AD) progression. A protease-resistant Cholecystokinin (CCK) analogue used in this study is based on the basic structure of CCK, which further increases the stability of the peptide fragment and prolongs its half-life in vivo. We observed a neuroprotective effect of CCK-8L in APPswe/PS1dE9 (APP/PS1) AD mice. However, its corresponding mechanisms still need to be elucidated. OBJECTIVE: This study examined CCK-8L's neuroprotective effects in enhancing cognitive impairment by regulating mitochondrial dynamics through AMPK/Drp1 pathway in the APP/PS1 AD mice. METHODS: Behavioural tests are applied to assess competence in cognitive functions. Transmission electron microscopy (TEM) was performed to observe the ultrastructure of mitochondria of hippocampal neurons, Immunofluorescent staining was employed to assay for Aß1-42, APP, Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and dynamin-related protein1 (Drp1). CRISPR/Cas9 was utilized for targeted knockout of the CCKB receptor (CCKBR) in the mouse APP/PS1 hippocampal CA1 region. A model of lentiviral vector-mediated overexpression of APP in N2a cells was constructed. RESULTS: In vivo, experiments revealed that CCK analogue and liraglutide significantly alleviated cognitive deficits in APP/PS1 mice, reduced Aß1-42 expression, and ameliorated l damage, which is associated with CCKBR activation in the hippocampal CA1 region of mice. In vitro tests showed that CCK inhibited mitochondrial fission and promoted fusion through AMPK/Drp1 pathway. CONCLUSIONS: CCK analogue ameliorates cognitive deficits and regulates mitochondrial dynamics by activating the CCKB receptor and the AMPK/Drp1 pathway in AD mice.

No link between handedness and spatial navigation: evidence from over 400 000 participants in 41 countries.

There is an active debate concerning the association of handedness and spatial ability. Past studies used small sample sizes. Determining the effect of handedness on spatial ability requires a large, cross-cultural sample of participants and a navigation task with real-world validity. Here, we overcome these challenges via the mobile app Sea Hero Quest. We analysed the navigation performance from 422 772 participants from 41 countries and found no reliable evidence for any difference in spatial ability between left- and right-handers across all countries. A small but growing gap in performance appears for participants over 64 years old, with left-handers outperforming right-handers. Further analysis, however, suggests that this gap is most likely due to selection bias. Overall, our study clarifies the factors associated with spatial ability and shows that left-handedness is not associated with either a benefit or a deficit in spatial ability.

Cultural determinants of the gap between self-estimated navigation ability and wayfinding performance: evidence from 46 countries.

Cognitive abilities can vary widely. Some people excel in certain skills, others struggle. However, not all those who describe themselves as gifted are. One possible influence on self-estimates is the surrounding culture. Some cultures may amplify self-assurance and others cultivate humility. Past research has shown that people in different countries can be grouped into a set of consistent cultural clusters with similar values and tendencies, such as attitudes to masculinity or individualism. Here we explored whether such cultural dimensions might relate to the extent to which populations in 46 countries overestimate or underestimate their cognitive abilities in the domain of spatial navigation. Using the Sea Hero Quest navigation test and a large sample (N = 383,187) we found cultural clusters of countries tend to be similar in how they self-rate ability relative to their actual performance. Across the world population sampled, higher self-ratings were associated with better performance. However, at the national level, higher self-ratings as a nation were not associated with better performance as a nation. Germanic and Near East countries were found to be most overconfident in their abilities and Nordic countries to be most under-confident in their abilities. Gender stereotypes may play a role in mediating this pattern, with larger national positive attitudes to male stereotyped roles (Hofstede's masculinity dimension) associated with a greater overconfidence in performance at the national level. We also replicate, with higher precision than prior studies, evidence that older men tend to overestimate their navigation skill more than other groups. These findings give insight into how culture and demographics may impact self-estimates of our abilities.

Entropy and a sub-group of geometric measures of paths predict the navigability of an environment.

Despite extensive research on navigation, it remains unclear which features of an environment predict how difficult it will be to navigate. We analysed 478,170 trajectories from 10,626 participants who navigated 45 virtual environments in the research app-based game Sea Hero Quest. Virtual environments were designed to vary in a range of properties such as their layout, number of goals, visibility (varying fog) and map condition. We calculated 58 spatial measures grouped into four families: task-specific metrics, space syntax configurational metrics, space syntax geometric metrics, and general geometric metrics. We used Lasso, a variable selection method, to select the most predictive measures of navigation difficulty. Geometric features such as entropy, area of navigable space, number of rings and closeness centrality of path networks were among the most significant factors determining the navigational difficulty. By contrast a range of other measures did not predict difficulty, including measures of intelligibility. Unsurprisingly, other task-specific features (e.g. number of destinations) and fog also predicted navigation difficulty. These findings have implications for the study of spatial behaviour in ecological settings, as well as predicting human movements in different settings, such as complex buildings and transport networks and may aid the design of more navigable environments.

Entropy of city street networks linked to future spatial navigation ability.

The cultural and geographical properties of the environment have been shown to deeply influence cognition and mental health1-6. Living near green spaces has been found to be strongly beneficial7-11, and urban residence has been associated with a higher risk of some psychiatric disorders12-14-although some studies suggest that dense socioeconomic networks found in larger cities provide a buffer against depression15. However, how the environment in which one grew up affects later cognitive abilities remains poorly understood. Here we used a cognitive task embedded in a video game16 to measure non-verbal spatial navigation ability in 397,162 people from 38 countries across the world. Overall, we found that people who grew up outside cities were better at navigation. More specifically, people were better at navigating in environments that were topologically similar to where they grew up. Growing up in cities with a low street network entropy (for example, Chicago) led to better results at video game levels with a regular layout, whereas growing up outside cities or in cities with a higher street network entropy (for example, Prague) led to better results at more entropic video game levels. This provides evidence of the effect of the environment on human cognition on a global scale, and highlights the importance of urban design in human cognition and brain function.

The National Landscape of Living Kidney Donor Follow-Up in the United States.

In 2013, the Organ Procurement and Transplantation Network (OPTN)/ United Network for Organ Sharing (UNOS) mandated that transplant centers collect data on living kidney donors (LKDs) at 6 months, 1 year, and 2 years postdonation, with policy-defined thresholds for the proportion of complete living donor follow-up (LDF) data submitted in a timely manner (60 days before or after the expected visit date). While mandated, it was unclear how centers across the country would perform in meeting thresholds, given potential donor and center-level challenges of LDF. To better understand the impact of this policy, we studied Scientific Registry of Transplant Recipients data for 31,615 LKDs between January 2010 and June 2015, comparing proportions of complete and timely LDF form submissions before and after policy implementation. We also used multilevel logistic regression to assess donor- and center-level characteristics associated with complete and timely LDF submissions. Complete and timely 2-year LDF increased from 33% prepolicy (January 2010 through January 2013) to 54% postpolicy (February 2013 through June 2015) (p < 0.001). In an adjusted model, the odds of 2-year LDF increased by 22% per year prepolicy (p < 0.001) and 23% per year postpolicy (p < 0.001). Despite these annual increases in LDF, only 43% (87/202) of centers met the OPTN/UNOS-required 6-month, 1-year, and 2-year LDF thresholds for LKDs who donated in 2013. These findings motivate further evaluation of LDF barriers and the optimal approaches to capturing outcomes after living donation.

Neuroprotective effects of lixisenatide and liraglutide in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.

Glucagon-like peptide 1 (GLP-1) is a growth factor. GLP-1 mimetics are on the market as treatments for type 2 diabetes and are well tolerated. These drugs have shown neuroprotective properties in animal models of neurodegenerative disorders. In addition, the GLP-1 mimetic exendin-4 has shown protective effects in animal models of Parkinson's disease (PD), and a clinical trial in PD patients showed promising first results. Liraglutide and lixisenatide are two newer GLP-1 mimetics which have a longer biological half-life than exendin-4. We previously showed that these drugs have neuroprotective properties in an animal model of Alzheimer's disease. Here we demonstrate the neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MPTP was injected once-daily (20mg/kg i.p.) for 7 days, and drugs were injected once-daily for 14 days i.p. When comparing exendin-4 (10 nmol/kg), liraglutide (25 nmol/kg) and lixisenatide (10 nmol/kg), it was found that exendin-4 showed no protective effects at the dose chosen. Both liraglutide and lixisenatide showed effects in preventing the MPTP-induced motor impairment (Rotarod, open-field locomotion, catalepsy test), reduction in tyrosine hydroxylase (TH) levels (dopamine synthesis) in the substantia nigra and basal ganglia, a reduction of the pro-apoptotic signaling molecule BAX and an increase in the anti-apoptotic signaling molecule B-cell lymphoma-2. The results demonstrate that in this study, both liraglutide and lixisenatide are superior to exendin-4, and both drugs show promise as a novel treatment of PD.

Lixisenatide rescues spatial memory and synaptic plasticity from amyloid ß protein-induced impairments in rats.

Alzheimer's disease (AD) is a progressive and degenerative disorder accompanied by cognitive impairment, but effective strategies against AD are currently not available. Interestingly, glucagon-like peptide-1 (GLP-1) used in type 2 diabetes mellitus (T2DM) has shown neuroprotective effects in preclinical studies of AD. Lixisenatide, an effective GLP-1 receptor (GLP-1R) agonist with much longer half life than GLP-1, has been licensed in the EU as a treatment for T2DM. However, the neuroprotective effects of lixisenatide in the brain remain to be clarified. In the present study, we report for the first time the effects of lixisenatide on the amyloid ß (Aß) protein-induced impairments in spatial learning and memory of rats, and investigated its electrophysiological and molecular mechanisms. We found that: (1) bilateral intrahippocampal injection of Aß25-35 resulted in a significant decline in spatial learning and memory of rats, as well as a suppression of in vivo hippocampal long-term potentiation (LTP); (2) lixisenatide treatment effectively prevented the Aß25-35-induced impairments; (3) lixisenatide inhibited the Aß25-35 injection-induced activation of glycogen synthase kinase 3ß (GSK3ß), with a significant increase in the phosphorylation of ser9 and a significant decrease in the phosphorylation of Y216. These results indicate that lixisenatide, by affecting the PI3K-Akt-GSK3ß pathway, can prevent Aß-related impairments in synaptic plasticity and spatial memory of rats, suggesting that lixisenatide may be a novel and effective treatment for AD.

Liraglutide improves hippocampal synaptic plasticity associated with increased expression of Mash1 in ob/ob mice.

OBJECTIVE: Consumption of high-fat diet exerts adverse effects on learning and memory formation, which is linked to impaired hippocampal function. Activation of glucagon-like peptide-1 (GLP-1) signalling ameliorates detrimental effects of obesity-diabetes on cognitive function; however, mechanisms underlying these beneficial actions remain unclear. This study examined effects of daily subcutaneous treatment with GLP-1 mimetic, Liraglutide, on synaptic plasticity, hippocampal gene expression and metabolic control in adult obese diabetic (ob/ob) mice. RESULTS: Long-term potentiation (LTP) induced by area CA1 was completely abolished in ob/ob mice compared with lean controls. Deleterious effects on LTP were rescued (P<0.001) with Liraglutide. Indeed, Liraglutide-treated mice exhibited superior LTP profile compared with lean controls (P<0.01). Expression of hippocampal brain-derived neurotropic factor and neurotrophic tyrosine kinase receptor-type 2 were not significantly different, but synaptophysin and Mash1 were decreased in ob/ob mice. Treatment with Liraglutide over 21 days increased expression of Mash1 in ob/ob mice (2.0-fold; P<0.01). These changes were associated with significantly reduced plasma glucose (21% reduction; P<0.05) and markedly improved plasma insulin concentrations (2.1- to 3.3-fold; P<0.05 to P<0.01). Liraglutide also significantly reduced the glycaemic excursion following an intraperitonal glucose load (area under curve (AUC) values: 22%; P<0.05) and markedly enhanced the insulin response to glucose (AUC values: 1.6-fold; P<0.05). O2 consumption, CO2 production, respiratory exchange ratio and energy expenditure were not altered by Liraglutide therapy. On day 21, accumulated food intake (32% reduction; P<0.05) and number of feeding bouts (32% reduction; P<0.05) were significantly reduced but simple energy restriction was not responsible for the beneficial actions of Liraglutide. CONCLUSION: Liraglutide elicits beneficial effects on metabolic control and synaptic plasticity in mice with severe obesity and insulin resistance mediated in part through increased expression of Mash1 believed to improve hippocampal neurogenesis and cell survival.

The incretin analogue D-Ala2GIP reduces plaque load, astrogliosis and oxidative stress in an APP/PS1 mouse model of Alzheimer's disease.

Type 2 diabetes mellitus has been identified as a risk factor for Alzheimer's disease (AD). Insulin is a neuroprotective growth factor, and an impairment of insulin signalling has been found in AD brains. Glucose-dependent insulinotropic polypeptide (GIP), an incretin hormone, normalises insulin signalling and also acts as a neuroprotective growth factor. GIP plays an important role in memory formation, synaptic plasticity and cell proliferation. We have shown previously that the long-lasting incretin hormone analogue D-Ala(2)GIP protects memory formation and synaptic plasticity, reduces plaques, normalises the proliferation of stem cells, reduces the activation of microglia, and prevents the loss of synapses in the cortex of the APPswe/PS1deltaE9 mouse model of Alzheimer's disease. D-Ala(2)GIP was injected for 35 days at 25 nmol/kg i.p. once daily in APP/PS1 male mice and wild-type (WT) littermates aged 6, 12 and 19 months. In a follow-up study, we analysed plaque load, the activation of astrocytes as a means of chronic inflammation in the brain, and oxidative stress in the brains of these mice (8-oxoguanine levels). D-Ala(2)GIP reduced the amyloid plaque load in 12- and 19-month-old mice, and the inflammation response as shown in the reduction of activated astrocytes in 12- and 19-month old APP/PS1 mice. Chronic oxidative stress in the brain was reduced in 12- and 19-month-old mice as shown in the reduction of 8-oxoguanine levels in the cortex of D-Ala(2)GIP-injected APP/PS1 mice. The results demonstrate that D-Ala(2)GIP has neuroprotective properties on key markers found in Alzheimer's disease. This finding shows that novel GIP analogues have the potential to be developed as novel therapeutics for Alzheimer's disease.

Novel GLP-1 mimetics developed to treat type 2 diabetes promote progenitor cell proliferation in the brain.

One of the symptoms of diabetes is the progressive development of neuropathies. One mechanism to replace neurons in the CNS is through the activation of stem cells and neuronal progenitor cells. We have tested the effects of the novel GLP-1 mimetics exenatide (exendin-4; Byetta) and liraglutide (NN2211; Victoza), which are already on the market as treatments for type 2 diabetes, on the proliferation rate of progenitor cells and differentiation into neurons in the dentate gyrus of brains of mouse models of diabetes. GLP-1 analogues were injected subcutaneously for 4, 6, or 10 weeks once daily in three mouse models of diabetes: ob/ob mice, db/db mice, or high-fat-diet-fed mice. Twenty-four hours before perfusion, animals were injected with 5'-bromo-2'-deoxyuridine (BrdU) to mark dividing progenitor cells. By using immunohistochemistry and stereological methods, the number of progenitor cells or doublecortin-positive young neurons in the dentate gyrus was estimated. We found that, in all three mouse models, progenitor cell division was enhanced compared with nondiabetic controls after chronic i.p. injection of either liraglutide or exendin-4 by 100-150% (P < 0.001). We also found an increase in young neurons in the DG of high-fat-diet-fed mice after drug treatment (P < 0.001). The GLP-1 receptor antagonist exendin(9-36) reduced progenitor cell proliferation in these mice. The results demonstrate that GLP-1 mimetics show promise as a treatment for neurodegenerative diseases such as Alzheimer's disease, because these novel drugs cross the blood-brain barrier and increase neuroneogenesis.

Four weeks administration of Liraglutide improves memory and learning as well as glycaemic control in mice with high fat dietary-induced obesity and insulin resistance.

AIM: Liraglutide is a long-acting glucagon-like peptide-1 (GLP-1) mimetic which is a treatment option for type 2 diabetes. GLP-1 peptides, including Liraglutide, cross the blood-brain barrier and may additionally act to improve brain function. The present study tested the hypothesis that, in addition to its antihyperglycaemic actions, peripheral administration of Liraglutide exerts positive actions on cognitive function in mice with high fat dietary-induced obesity and insulin resistance. METHODS: Young Swiss TO mice maintained on high fat diet for 20 weeks received twice-daily injections of Liraglutide (200 µg/kg bw; sc) or saline vehicle over 28 days. An additional group of mice on standard diet received twice-daily saline injections. Energy intake, bodyweight, non-fasting plasma glucose and insulin concentrations were monitored at regular intervals. Glucose tolerance, open field assessment, object recognition testing and electrophysiological long-term potentiation (LTP) were performed at termination of the study. RESULTS: Liraglutide treatment resulted in significant time-dependent reduction in bodyweight and energy intake, whilst improving non-fasting glucose and normalizing glucose tolerance. Although Liraglutide did not alter general behaviour, treated mice exhibited marked increase in recognition index (RI) during object recognition testing, indicative of enhanced learning and memory ability. Furthermore, Liraglutide rescued the deleterious effects of high fat diet on hippocampal LTP of neurotransmission following both chronic and direct intracerebroventricular (icv) administration. CONCLUSION: Liraglutide administered peripherally not only improves metabolic parameters but exerts additional beneficial effects on cognitive function and hippocampal synaptic plasticity. Whether therapy with GLP-1 mimetics has similar effects in humans with type 2 diabetes needs to be established.

Val8-glucagon-like peptide-1 protects against Aß1-40-induced impairment of hippocampal late-phase long-term potentiation and spatial learning in rats.

Amyloid ß protein (Aß) is considered to be partly responsible for the impairment of learning and memory in Alzheimer disease (AD). In addition, it has been found recently that type 2 diabetes mellitus (T2DM) is a risk factor for developing AD. One promising treatment for AD is using analogues for the insulin-release facilitating gut hormone glucagon-like peptide-1 (GLP-1) that has been developed as a T2DM therapy. GLP-1 has been shown to have neuroprotective properties. However, if GLP-1 can protect the late phase-long term potentiation (L-LTP) and related cognitive function against Aß-induced impairment it is still an open question. To further characterize the neuroprotective function of GLP-1 in the brain, we investigated the effects of i.c.v. injected Val(8)-GLP-1(7-36) on the Aß fragment-induced impairment of in vivo hippocampal L-LTP and spatial learning and memory in rats. The results showed that (1) Aß1-40 (5 nmol) injection did not affect the baseline field excitatory postsynaptic potentials (fEPSPs), but significantly suppressed multiple high frequency stimulation (HFS)-induced L-LTP in hippocampal CA1 region; (2) Val(8)-GLP-1(7-36) (0.05 pmol) administration alone did not affect the baseline synaptic transmission and the maintenance of L-LTP; (3) pretreatment with Val(8)-GLP-1(7-36) (0.05 pmol) effectively prevented Aß1-40-induced deficit of L-LTP; (4) i.c.v. injection of 5 nmol Aß1-40 resulted in a significant decline learning a spatial Morris water maze (MWM) test; (5) Val(8)-GLP-1(7-36) (0.05 pmol) administration alone did not affect spatial learning in this task, while pretreatment with Val(8)-GLP-1(7-36) effectively reversed the impairment of spatial learning and memory induced by Aß1-40. At the same time, the swim speeds and escape latencies of rats among all groups in the visible platform tests did not show any difference. These results suggest that increase of GLP-1 signalling in the brain may be a promising strategy to ameliorate the degenerative processes observed in AD.

Actions of exendin-4 therapy on cognitive function and hippocampal synaptic plasticity in mice fed a high-fat diet.

High-calorie diet has been shown to impair learning ability and hippocampal synaptic plasticity in rodents. This study examined effects of daily treatment with the glucagon-like peptide-1 mimetic, exendin-4, on cognitive function and hippocampal synaptic plasticity in a model of diet-induced obesity, which exhibits compromised cognitive performance. Mice fed a high-fat diet were treated with exendin-4 (25 nmol kg(-1) bodyweight; twice daily) or saline vehicle (0.9% (w/v) NaCl) over 21 days. In addition to improving metabolic control, exendin-4-treated mice exhibited a marked increase in recognition index highlighting improved learning and memory. High-fat diet resulted in the elimination of in vivo electrophysiological long-term potentiation, which was rescued following exendin-4 treatment. This study shows that exendin-4 therapy improves cognitive function and ameliorates impaired hippocampal synaptic plasticity in dietary-induced obesity.

The effect of mGlu8 deficiency in animal models of psychiatric diseases.

The metabotropic glutamate receptor subtype 8 (mGlu(8)) is presynaptically located and regulates the release of the transmitter. Dysfunctions of this mechanism are involved in the pathophysiology of different psychiatric disorders. mGlu(8) deficient mice have been previously investigated in a range of studies, but the results are contradictory and there are still many open questions. Therefore, we tested mGlu(8)-deficient animals in different behavioral tasks that are commonly used in neuropsychiatric research. Our results show a robust contextual fear deficit in mGlu(8)-deficient mice. Furthermore, novel object recognition, chlordiazepoxide-facilitated extinction of operant conditioning and the acoustic startle response were attenuated by mGlu(8) deficiency. We found no changes in sensory processing, locomotor activity, prepulse inhibition, phencyclidine-induced changes in locomotion or prepulse inhibition, operant conditioning, conditioned fear to a discrete cue or in animal models of innate fear and post-traumatic stress disorder. We conclude that mGlu(8) might be a potential target for disorders with pathophysiological changes in brain areas where mGlu(8) modulates glutamate and gamma-amino butyric acid (GABA) transmission. Our data especially point to anxiety disorders involving exaggerated contextual fear, such as generalized anxiety disorders, and to conditions with disturbed declarative memory.

LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement.

Tuberculosis control relies on the identification and preventive treatment of individuals who are latently infected with Mycobacterium tuberculosis. However, direct identification of latent tuberculosis infection is not possible. The diagnostic tests used to identify individuals latently infected with M. tuberculosis, the in vivo tuberculin skin test and the ex vivo interferon-gamma release assays (IGRAs), are designed to identify an adaptive immune response against, but not necessarily a latent infection with, M. tuberculosis. The proportion of individuals who truly remain infected with M. tuberculosis after tuberculin skin test or IGRA conversion is unknown. It is also uncertain how long adaptive immune responses towards mycobacterial antigens persist in the absence of live mycobacteria. Clinical management and public healthcare policies for preventive chemotherapy against tuberculosis could be improved, if we were to gain a better understanding on M. tuberculosis latency and reactivation. This statement by the TBNET summarises knowledge and limitations of the currently available tests used in adults and children for the diagnosis of latent tuberculosis infection. In summary, the main issue regarding testing is to restrict it to those who are known to be at higher risk of developing tuberculosis and who are willing to accept preventive chemotherapy.

Rats are able to navigate in virtual environments.

Virtual reality (VR) systems are useful tools that enable users to alter environmental settings and the location of landmarks in an accurate and fast way. Primates have been shown to be able to navigate in virtual environments. For rodents, however, all previous attempts to develop VR systems in which rats behave in the same way as in corresponding 3-D environments have failed. The question arises as to whether, in principle, rodents can be trained to navigate in a properly designed virtual environment (VE), or whether this peculiarity is limited to primates and humans. We built a virtual reality set-up that takes the wide-angle visual system of rats into account. We show for the first time that rats learn spatial tasks in this VE quite readily. This set-up opens up new opportunities for investigations of information processing in navigation (e.g. the importance of optic flow or vestibular input).

Effects of the isolation methodology on protein profiles of blood trypomastigotes of Trypanosoma cruzi.

Blood trypomastigotes of Trypanosoma cruzi were isolated from infected athymic rnu/rnu rats and purified by an improved procedure of DEAE-Sephacel ion-exchange chromatography. Elution into a buffer supplemented with bovine serum albumin avoided column-induced changes on the surface of the parasites. Biotin-labelled bovine serum albumin, fluorescence microscopy, flow cytometry and Western blot analysis revealed a very intense binding of albumin to the parasite. Incubation and washing of cells without protein supplementation did not result in any damage or lysis of parasites but it did cause extensive shedding of cellular and surface proteins into the supernatant which could be prevented by using the protein-supplemented buffer. A decreasing yield of high molecular weight cellular proteins in relation to centrifugal force was a general phenomenon observed in scanning densitometry of SDS gels after isolation in either protein-supplemented buffer or protein-free buffer. The quantity of shed cellular components increased with increasing centrifugal force. In contrast, quantities of high molecular weight, biotin-labelled surface proteins increased with greater centrifugal force, indicating labelling of otherwise inaccessible residues. These data emphasize the importance of protein supplementation of buffers with proteins and of choosing low centrifugation forces (<400 g) during investigations of T. cruzi.

A protective and agonistic function of IL-12p40 in mycobacterial infection.

IL-12p35(-/-)p40(-/-) mice are highly susceptible to Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis infection. In this study IL-12p35(-/-) mice, which are able to produce endogenous IL-12p40, cleared M. bovis BCG and showed reduced susceptibility to pulmonary M. tuberculosis infection, which was in striking contrast to the outcome of mycobacterial infection in IL-12p35(-/-)p40(-/-) mice. Resistance in wild-type and IL-12p35(-/-) mice was accompanied by protective granuloma formation and Ag-specific delayed-type hypersensitivity responses, which were impaired in susceptible IL-12p35(-/- )p40(-/-) mice. Furthermore, IL-12p35(-/-) mice, but not IL-12p35(-/-)p40(-/-) mice, mounted Ag-specific Th1 and cytotoxic T cell responses. In vivo therapy with rIL-12p40 homodimer restored the impaired delayed-type hypersensitivity responses in M. bovis BCG-infected IL-12p35(-/-)p40(-/-) mice and reverted them to a more resistant phenotype. Together, these results show evidence for a protective and agonistic role of endogenous and exogenous IL-12p40 in mycobacterial infection, which is independent of IL-12p70.

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.