Purification of cytoplasmic antigens from the mycelial phase of Candida albicans: Possible advantages of its use in Candida serology

The serology of candidiasis is complicated by the use of poorly defined antigens. Total extracts of the yeast phase have been commonly used as cytoplasmic antigen, without regard to the significant amounts of carbohydrate that may contaminate such preparations. This is particularly true in the case of commercially available antigens that have been used as cytoplasmic antigens but actually are richer in carbohydrate than in protein. Affinity chromatography in concanavalin A — Sepharose provides a simple procedure to separate carbohydrates, mainly mannan, from protein antigens in whole Candida extracts. By using mannan-poor antigens, the specificity of serological reactions can be increased considerably, since both the positive reactions seen in asymptomatic donors and the cross-reactions seen in patients infected with other fungi are due to anti-mannan antibodies. In contrast, both anti-mannan and anti-cytoplasmic antigen antibodies can be detected in patients suspected of systemic candidiasis. On the other hand, absolute specificity may never be achieved for systemic candidiasis. We have found antibodies against cytoplasmic antigen in a patient allergic to C. albicans, in whom the microorganism was isolated from fecal material. It appears that, under favorable conditions, mucosal sensitization may also trigger a systemic reaction directed against both mannan and cytoplasmic antigens.Publication no. 341 from The Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina.     

Sesquiterpene lactones of Podanthus mitiqui

Ovatifolin and two new sesquiterpene lactones, deacetylovatifolin and arturin (1β-hydroxy-8β-angeloyloxy-eudesmane-4(15),11(13)-diene-6α,12-olide, have been isolated from stems and leaves of Podanthus mitiqui. Two of these compounds showed cytotoxic activity.     

Patent Human Infections with the Whipworm,Trichuris trichiura,Are Not Associated with Alterations in the Faecal Microbiota

Background

The soil-transmitted helminth (STH), Trichuris trichiura colonises the human large intestine where it may modify inflammatory responses, an effect possibly mediated through alterations in the intestinal microbiota. We hypothesised that patent T. trichiura infections would be associated with altered faecal microbiota and that anthelmintic treatment would induce a microbiota resembling more closely that observed in uninfected individuals.

Materials and Methods

School children in Ecuador were screened for STH infections and allocated to 3 groups: uninfected, T. trichiura only, and mixed infections with T. trichiura and Ascaris lumbricoides. A sample of uninfected children and those with T. trichiura infections only were given anthelmintic treatment. Bacterial community profiles in faecal samples were studied by 454 pyrosequencing of 16 S rRNA genes.

Results

Microbiota analyses of faeces were done for 97 children: 30 were uninfected, 17 were infected with T. trichiura, and 50 with T. trichiura and A. lumbricoides. Post-treatment samples were analyzed for 14 children initially infected with T. trichiura alone and for 21 uninfected children. Treatment resulted in 100% cure of STH infections. Comparisons of the microbiota at different taxonomic levels showed no statistically significant differences in composition between uninfected children and those with T. trichiura infections. We observed a decreased proportional abundance of a few bacterial genera from the Clostridia class of Firmicutes and a reduced bacterial diversity among children with mixed infections compared to the other two groups, indicating a possible specific effect of A. lumbricoides infection. Anthelmintic treatment of children with T. trichiura did not alter faecal microbiota composition.

Discussion

Our data indicate that patent human infections with T. trichiura may have no effect on faecal microbiota but that A. lumbricoides colonisation might be associated with a disturbed microbiota. Our results also catalogue the microbiota of rural Ecuadorians and indicate differences with individuals from more urban industrialised societies.     

Leukemic histiocytic sarcoma in a captive common squirrel monkey (Saimiri sciureus) with Saimiriine Gammaherpesvirus 2 (Rhadinovirus), Saimiri sciureus lymphocryptovirus 2 (Lymphocryptovirus) and Squirrel monkey retrovirus (β-Retrovirus) coinfection

Hematopoietic neoplasia other than lymphoma and leukemia is uncommon among non-human primates. Herein, we provide the first evidence of occurrence of leukemic histiocytic sarcoma in a captive common squirrel monkey with Saimiriine Gammaherpesvirus 2 (Rhadinovirus), Saimiri sciureus lymphocryptovirus 2 (Lymphocryptovirus), and Squirrel monkey retrovirus (β-Retrovirus) coinfection.     

Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A

The cAMP-dependent protein kinase (PKA) is the best understood member of the superfamily of serine–threonine protein kinases and is involved in controlling a variety of cellular processes. Measurements of PKA activity traditionally relied on the use of [³²P]-labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. Recently non-isotopic assays for the PKA have been developed and this paper presents an improvement of a fluorometric assay for measuring the activity of PKA. Three peptides were synthesized with the following sequences: LRRASLG (Kemptide), LRRASLGK (Kemptide-Lys8) and LRRASLGGGLRRASLG (Bis-Kemptide), these have in common the substrate sequence recognized by the PKA (RRXS/TΨ), where X is any amino acid and Ψ is a hydrophobic amino acid. Optimal conditions were established for the non-radioactive assay to detect the PKA activity by phosphorylation of these three peptides that are covalently linked to fluorescamine at their N-terminus. The phosphorylated and non-phosphorylated peptides were easily separated by electrophoresis, identified and quantified with optical densitometry and ultraviolet light. The fluorescamine-labeled Kemptide-Lys8 substrate (Fluram-Kemptide-Lys8) was used to calculate the Km and V_max of the catalytic subunit of PKA from pig heart and showed a detection limit of 260 pmol, a linear range between 700 and 1150 pmol with a linear regression R ² = 0.956.     

Hemozoin increases IFN-gamma-inducible macrophage nitric oxide generation through extracellular signal-regulated kinase- and NF-kappa B-dependent pathways

NO overproduction has been suggested to contribute to the immunopathology related to malaria infection. Even though a role for some parasite molecules (e.g., GPI) in NO induction has been proposed, the direct contribution of hemozoin (HZ), another parasite metabolite, remains to be established. Therefore, we were interested to determine whether Plasmodium falciparum (Pf) HZ and synthetic HZ, beta-hematin, alone or in combination with IFN-gamma, were able to induce macrophage (Mphi) NO synthesis. We observed that neither Pf HZ nor synthetic HZ led to NO generation in B10R murine Mphi; however, they significantly increased IFN-gamma-mediated inducible NO synthase (iNOS) mRNA and protein expression, and NO production. Next, by investigating the transductional mechanisms involved in this cellular regulation, we established that HZ induces extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase phosphorylation as well as NF-kappaB binding to the iNOS promoter, and enhances the IFN-gamma-dependent activation of both second messengers. Of interest, cell pretreatment with specific inhibitors against either NF-kappaB or the ERK1/2 pathway blocked the HZ + IFN-gamma-inducible NF-kappaB activity and significantly reduced the HZ-dependent increase on IFN-gamma-mediated iNOS and NO induction. Even though selective inhibition of the Janus kinase 2/STAT1alpha pathway suppressed NO synthesis in response to HZ + IFN-gamma, HZ alone did not activate this signaling pathway and did not have an up-regulating effect on the IFN-gamma-induced Janus kinase 2/STAT1alpha phosphorylation and STAT1alpha binding to the iNOS promoter. In conclusion, our results suggest that HZ exerts a potent synergistic effect on the IFN-gamma-inducible NO generation in Mphi via ERK- and NF-kappaB-dependent pathways.     

A reversible abnormal form of myelin: an X-ray scattering study of human sural and rat sciatic nerves

A sural nerve dissected from a recently dead patient displayed an unusual X-ray diffraction pattern, suggesting that in situ and at the time of the patient's death the myelin sheaths were in a swollen state. Diffraction patterns of the swollen type were also recorded from: (1) a sural nerve from the corpse of a neurologically healthy person after soaking the nerve with Ringer solution at pH 5.5; (2) sciatic nerves dissected from rat cadavers at increasing time after death. In all the cases the swollen patterns reversed to the native type upon superfusion with Ringer solution at pH 7.3. The postmortem effect is to decrease the pH of the fluids surrounding the nerves in the cadavers. Our experiments show that the early postmortem processes have the effect of acidifying PNS nerves and that as a consequence of acidification the myelin sheaths swell.     

Effects of Hormone Therapy on Cognition and Mood in Recently Postmenopausal Women: Findings from the Randomized,Controlled KEEPS–Cognitive and Affective Study

Background

Menopausal hormone therapy (MHT) reportedly increases the risk of cognitive decline in women over age 65 y. It is unknown whether similar risks exist for recently postmenopausal women, and whether MHT affects mood in younger women. The ancillary Cognitive and Affective Study (KEEPS-Cog) of the Kronos Early Estrogen Prevention Study (KEEPS) examined the effects of up to 4 y of MHT on cognition and mood in recently postmenopausal women.

Methods and Findings

KEEPS, a randomized, double-blinded, placebo-controlled clinical trial, was conducted at nine US academic centers. Of the 727 women enrolled in KEEPS, 693 (95.3%) participated in the ancillary KEEPS-Cog, with 220 women randomized to receive 4 y of 0.45 mg/d oral conjugated equine estrogens (o-CEE) plus 200 mg/d micronized progesterone (m-P) for the first 12 d of each month, 211 women randomized to receive 50 μg/d transdermal estradiol (t-E2) plus 200 mg/d m-P for the first 12 d of each month, and 262 women randomized to receive placebo pills and patches. Primary outcomes included the Modified Mini-Mental State examination; four cognitive factors: verbal learning/memory, auditory attention/working memory, visual attention/executive function, and speeded language/mental flexibility; and a mood measure, the Profile of Mood States (POMS). MHT effects were analyzed using linear mixed-effects (LME) models, which make full use of all available data from each participant, including those with missing data. Data from those with and without full data were compared to assess for potential biases resulting from missing observations. For statistically significant results, we calculated effect sizes (ESs) to evaluate the magnitude of changes.On average, participants were 52.6 y old, and 1.4 y past their last menstrual period. By month 48, 169 (24.4%) and 158 (22.8%) of the 693 women who consented for ancillary KEEPS-Cog were lost to follow-up for cognitive assessment (3MS and cognitive factors) and mood evaluations (POMS), respectively. However, because LME models make full use all available data, including data from women with missing data, 95.5% of participants were included in the final analysis (n = 662 in cognitive analyses, and n = 661 in mood analyses). To be included in analyses, women must have provided baseline data, and data from at least one post-baseline visit. The mean length of follow-up was 2.85 y (standard deviation [SD] = 0.49) for cognitive outcomes and 2.76 (SD = 0.57) for mood outcomes. No treatment-related benefits were found on cognitive outcomes. For mood, model estimates indicated that women treated with o-CEE showed improvements in depression and anxiety symptoms over the 48 mo of treatment, compared to women on placebo. The model estimate for the depression subscale was −5.36 × 10⁻² (95% CI, −8.27 × 10⁻² to −2.44 × 10^−2; ES = 0.49, p < 0.001) and for the anxiety subscale was −3.01 × 10⁻² (95% CI, −5.09 × 10⁻² to −9.34 × 10⁻³; ES = 0.26, p < 0.001). Mood outcomes for women randomized to t-E2 were similar to those for women on placebo. Importantly, the KEEPS-Cog results cannot be extrapolated to treatment longer than 4 y.

Conclusions

The KEEPS-Cog findings suggest that for recently postmenopausal women, MHT did not alter cognition as hypothesized. However, beneficial mood effects with small to medium ESs were noted with 4 y of o-CEE, but not with 4 y of t-E2. The generalizability of these findings is limited to recently postmenopausal women with low cardiovascular risk profiles.

Trial Registration

ClinicalTrials.gov NCT00154180 and NCT00623311     

Using Satellite Tracking and Isotopic Information to Characterize the Impact of South American Sea Lions on Salmonid Aquaculture in Southern Chile

Apex marine predators alter their foraging behavior in response to spatial and/or seasonal changes in natural prey distribution and abundance. However, few studies have identified the impacts of aquaculture that represents a spatially and temporally predictable and abundant resource on their foraging behavior. Using satellite telemetry and stable isotope analysis we examined the degree of spatial overlap between the South American sea lion (SASL) and salmon farms, and quantify the amount of native prey versus farmed salmonids in SASL diets. We instrumented eight SASL individuals with SRDL-GPS tags. Vibrissae, hair and skin samples were collected for δ¹³C and δ¹⁵N analyses from five of the tagged individuals and from four males captured in a haul-out located adjacent to salmon farms. Tracking results showed that almost all the foraging areas of SASL are within close proximity to salmon farms. The most important prey for the individuals analyzed was farmed salmonids, with an estimated median (±SD) contribution of 19.7 ± 13.5‰ and 15.3 ± 9.6‰ for hair and skin, respectively. Using vibrissae as a temporal record of diet for each individual, we observed a remarkable switch in diet composition in two SASL, from farmed salmonids to pelagic fishes, which coincided with the decrease of salmon production due to the infectious salmon anemia virus that affected salmon farms in Chile at the end of 2008. Our study demonstrates the usefulness of integrating stable isotope derived dietary data with movement patterns to characterize the impacts of a non-native prey on the foraging ecology of an apex marine predator, providing important applied implications in situations where interactions between aquaculture and wildlife are common.     

Boekbespreking

Frits J. Huyse, Friedrich C. Stiefel (eds.) Integrated care for the complex medically ill. Medical Clinics of North America. 2006. Vol. 90, Number 4.