Neuroprotective peptides influence cytokine and chemokine alterations in a model of fetal alcohol syndrome.

OBJECTIVE: Fetal alcohol syndrome (FAS) is associated with intellectual disability and neurodevelopmental abnormalities. Neuroprotective peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL) can prevent some of the alcohol-induced teratogenesis including fetal death, growth abnormalities, and learning impairment in part by preventing alcohol-induced alterations in N-methyl-D-aspartate receptor gene expression in a mouse model for FAS. We evaluated a panel of cytokines and chemokines to determine whether NAP plus SAL work through a cytokine/chemokine-mediated pathway in preventing these alterations. STUDY DESIGN: Using a well-characterized FAS model, timed, pregnant C57BL6/J mice were treated on gestational day (E) 8 with alcohol (0.03 mL/g), placebo, or alcohol plus peptides. Embryos were evaluated at 2 time points: after 6 hours and 10 days later at E18. A panel of cytokines/chemokines was measured using a microsphere-based multiplex immunoassay (Luminex xMAP; Millipore, Billerica, MA). Statistical analysis included Kruskal-Wallis, with P < .05 considered significant. RESULTS: Six hours after treatment, interleukin (IL)-6 and keratinocyte chemoattractant cytokine (KC) were not detectable in the control embryos. Alcohol treatment resulted in detectable levels and significant increases in IL-6 (median, 15.7; range, 10.1-45.9 pg/mL) and KC (median, 45.9; range, 32.5-99.1 pg/mL). Embryos exposed to alcohol plus NAP plus SAL had undetectable IL-6 and KC (both P < .003), similar to the controls. Alcohol exposure resulted in a significant increase of granulocyte colony-stimulating factor (G-CSF) (P < .003) as compared with controls, and treatment with NAP plus SAL prevented the alcohol-induced increase. IL-13 and IL-1ß were decreased 6 hours after alcohol exposure, and exposure to alcohol plus NAP plus SAL did not completely ameliorate the decrease. At E18, 10 days after exposure, these alterations were no longer present. Several analytes (regulated upon activation, normal T cell expressed, and secreted, tumor necrosis factor-α, interferon-γ, and IL-4) were not detectable at either time point in any of the groups. CONCLUSION: Prenatal alcohol exposure acutely results in a significant elevation of IL-6, G-CSF and the KC, which are known to affect N-methyl-D-aspartate receptors. NAP plus SAL treatment prevented alcohol-induced increases. This provides additional insight into the mechanism of alcohol damage in FAS and NAP plus SAL prevention of neurodevelopmental anomalies.

Cytokine and chemokine alterations in Down syndrome.

OBJECTIVE: Down syndrome (DS) is the leading genetic cause of intellectual disability, affecting ~1/800 newborns. Previously we have shown alterations in N-methyl-D-aspartic acid (NMDA) receptors and neuropeptides (activity-dependant neuroprotective protein, glia fibrillary acidic protein) in a murine model of DS. Cytokines and chemokines have neuromodulatory and neurotransmitter roles and interact with the NMDA receptors. The objective of this study was to evaluate if cytokines and chemokines in the hippocampus and cerebellum are altered in this model. STUDY DESIGN: We used 8- to 10-month-old animals from the well-characterized mouse model of DS (Ts65Dn). Learning and memory were assessed in the Morris water maze with the Ts65Dn animals demonstrating a learning deficit. After completion of the behavioral testing, the brains were removed and the hippocampus and cerebellum were separated by microdissection. A panel of cytokines, chemokines, and fractalkine were measured in the protein lysates using a microsphere-based multiplex immunoassay (Luminex xMAP, Millipore) and normalized to total protein concentration. Statistical analysis included the nonparametric Mann-Whitney U for the cytokine, chemokine, and fractalkine levels; p < 0.05 was considered significant. RESULTS: Levels (median [range]) of interleukin (IL)-1ß (6.95 [0.11 to 43.5] versus 14.2 [0.2 to 36.8] pg/mL); granulocyte-macrophage colony-stimulating factor (GM-CSF; 3.97 [0.19 to 19.6] versus 19.2 [0.2 to 31.1] pg/mL), and macrophage inflammatory protein (MIP)-1α (20.3 [0.11 to 73.3] versus 37.0 [0.22 to 102.7] pg/mL) in the hippocampus from Ts65Dn were significantly lower compared with the euploid (control) animals. Many cytokines and chemokines were not detected in the hippocampus or cerebellum, and others were detectable but not different between the groups. CONCLUSION: We found a decreased in GM-CSF, IL-1ß, and MIP-1α in the hippocampus of DS pups. All three have known interactions with NMDA receptors and their decline may explain, in part, the learning deficits associated with DS.

Strong viremia control in vaccinated macaques does not prevent gradual Th17 cell loss from central memory.

It has been proposed that microbial translocation might play a role in chronic immune activation during HIV/SIV infection. Key roles in fighting bacterial and fungal infections have been attributed to Th17 and Tc17 cells. Th17 cells can be infected with HIV/SIV, however whether effective vaccination leads to their maintenance following viral challenge has not been addressed. Here we retrospectively investigated if a vaccine regimen that potently reduced viremia post-challenge preserved Th17 and Tc17 cells, thus adding benefit in the absence of sterilizing protection. Rhesus macaques were previously vaccinated with replication-competent Adenovirus recombinants expressing HIVtat and HIVenv followed by Tat and gp140 protein boosting. Upon SHIV(89.6P) challenge, the vaccines exhibited a significant 4 log reduction in chronic viremia compared to sham vaccinated controls which rapidly progressed to AIDS [39]. Plasma and cryopreserved PBMC samples were examined pre-challenge and during acute and chronic infection. Control macaques exhibited a rapid loss of CD4(+) cells, including Th17 cells. Tc17 cells tended to decline over the course of infection although significance was not reached. Immune activation, assessed by Ki-67 expression, was associated with elevated chronic viremia of the controls. Significantly increased plasma IFN-γ levels were also observed. No increase in plasma LPS levels were observed suggesting a lack of microbial translocation. In contrast, vaccinated macaques had no evidence of immune activation within the chronic phase and preserved both CD4(+) T-cells and Tc17 cells in PBMC. Nevertheless, they exhibited a gradual, significant loss of Th17 cells which concomitantly displayed significantly higher CCR6 expression over time. The gradual Th17 cell decline may reflect mucosal homing to inflammatory sites and/or slow depletion due to ongoing low levels of SHIV replication. Our results suggest that potent viremia reduction during chronic SHIV infection will delay but not prevent the loss of Th17 cells.

Neuroprotective fractalkine in fetal alcohol syndrome.

OBJECTIVE: Neuroprotective peptides (NAP+SAL) can prevent some alcohol-induced damage in fetal alcohol syndrome(FAS). Fractalkine, a chemokine constitutively expressed in the CNS reduces neuronal death from activated microglia. Using a model of FAS we evaluated if fractalkine is altered and if NAP+SAL work through fractalkine. STUDY DESIGN: Using a FAS model, C57BL6/J-mice were treated on gestational day 8 with alcohol (0.03 mL/g), placebo or alcohol+peptides. Embryos were harvested after 6h(E8) and 10 days later(E18). Fractalkine was measured in the protein lysate (Luminex xMAP). Statistical analysis included Kruskal-Wallis. RESULTS: Fractalkine was significantly elevated at 6h (median 341 pg/ml, range 263-424 pg/ml) vs. controls (median 228 pg/ml, range 146-332 pg/ml; P<.001). NAP+SAL prevented the alcohol-induced increase (median 137, range 97-255 pg/ml, P<.001). At E18, fractalkine levels were similar in all groups (P=0.7). CONCLUSION: Prenatal alcohol exposure acutely elevates fractalkine, perhaps in an effort to counter the alcohol toxicity. Pre-treatment with NAP+SAL prevents the acute increase in fractalkine.

Distinct clinical and immunologic profiles in severe malarial anemia and cerebral malaria in Zambia.

BACKGROUND: The mechanisms of severe malarial anemia and cerebral malaria, which are extreme manifestations of Plasmodium falciparum malaria, are not fully understood. METHODS: Children aged <6 years from southern Zambia presenting to the hospital with severe malarial anemia (n = 72), cerebral malaria (n = 28), or uncomplicated malaria (n = 66) were studied prospectively. Children with overlapping severe anemia and cerebral malaria were excluded. RESULTS: Low interleukin 10 concentrations had the strongest association with severe anemia (standard ß = .61; P < .001) followed by high tumor necrosis factor α and sFas concentrations, low weight-for-age z scores, presence of stool parasites, and splenomegaly (standard ß = .15-.25; P ≤ .031); most of these factors were also associated with lower reticulocytes. Greater parasitemia was associated with higher interleukin 10 and tumor necrosis factor α concentrations, whereas sulfadoxizole/pyrimethamine therapy and lower weight-for-age z scores were associated with lower interleukin 10 levels. Thrombocytopenia and elevated tissue plasminogen activator inhibitor 1 levels had the strongest associations with cerebral malaria (standard ß = .37 or .36; P < .0001), followed by exposure to traditional herbal medicine and hemoglobinuria (standard ß = .21-.31; P ≤ .006). CONCLUSIONS: Predictors of severe malarial anemia (altered immune responses, poor nutrition, intestinal parasites, and impaired erythropoiesis) differed from those of cerebral malaria (thrombocytopenia, herbal medicine, and intravascular hemolysis). Improved preventive and therapeutic measures may need to consider these differences.

Measurement of CGRP in dried blood spots using a modified sandwich enzyme immunoassay.

Calcitonin gene-related peptide (CGRP) has roles as a neurotransmitter, neuromodulator and trophic factor. CGRP has been reported to be elevated in neonatal blood of children with autism or mental retardation as compared with normal subjects by recycling immuno-affinity chromatography (RIC). While CGRP detection in neonatal blood is thus important, it is not easy to detect CGRP in dried blood spots because of the limitations of sample volume and the specificity and the sensitivity of available assay systems. In the present study, we modified a "Sandwich Enzyme Immunoassay" for the purpose of detecting CGRP in blood spot eluate. We have prepared blood spots from blood collected from normal human subjects and measured CGRP level in eluates from these blood spots. Instead of a purification step, we have introduced a pre-incubation step and used washed erythrocytes as a dilution solution. The modified assay has good recovery and specificity and appropriate dilution curves. We have compared the eluate levels with levels in serum and plasma from the same individuals and find that CGRP levels in blood spot eluate were similar to those of serum and plasma. Thus, the newly modified EIA may be a useful method for the detection of CGRP in blood spot eluate.

Selected neurotrophins, neuropeptides, and cytokines: developmental trajectory and concentrations in neonatal blood of children with autism or Down syndrome.

Using a double-antibody immunoaffinity assay (Luminex) and ELISA technology, we measured concentrations of certain neurotrophins, neuropeptides, and cytokines in pooled samples (one to three subjects per sample) eluted from archived neonatal blood of children with later-diagnosed autism, Down syndrome, very preterm birth, or term control infants. We also measured analytes in blood from healthy adult controls. Case or control status for infant subjects was ascertained by retrospective review of service agency medical records. We observed inhibitory substances in eluates from archived bloodspots, especially marked for measurement of BDNF. Concentrations in control subjects differed by age: BDNF rose markedly with age, while NT-3 and NT-4/5 concentrations were lower in adults than in newborn infants. IL-8 concentrations were higher in newborn infants, preterm and term, than in adults. Considered by diagnostic group, total protein was higher in Down syndrome than in either autism or control subjects. In infants with Down syndrome, concentrations of IL-8 levels were higher than in controls, whether or not corrected for total protein; NT-3 and CGRP were lower and VIP higher. In samples from autistic subjects, NT-3 levels were significantly lower than controls and an increase in VIP approached statistical significance. Concentrations of NT-4/5 and CGRP were correlated in infants with autism but not in Down syndrome or controls. Some of these results differ from earlier findings using a single-antibody recycling immunoaffinity chromatography (RIC) system. We discuss interrelationships of VIP, NT-3 and IL-8 and their potential relevance to features of the neuropathology of autism or Down syndrome.

Measurement of vasoactive intestinal peptide using a competitive fluorescent microsphere immunoassay or ELISA in human blood samples.

The concentration of Vasoactive Intestinal Peptide (VIP) as measured by recycling immunoaffinity chromatography (RIC) has been reported to be elevated in the blood of patients with autism as compared with normal subjects. In this study, we have developed a "Competitive Fluorescent Microsphere Immunoassay" (cFMI) in which VIP competes with biotinylated VIP in binding to polyclonal antibodies on microspheres. The results were obtained using the Luminex100 system. We measured VIP in serum, plasma, and material eluted from dried blood spots on filter paper with both the cFMI and an ELISA procedure. We found that a purification procedure was necessary for obtaining useful results from plasma and serum, however, a preincubation step was required with the blood eluates. This newly developed cFMI was more sensitive (2.5 vs. 20.0 pg/ml), and more reproducible than the ELISA. To get accurate measurements of VIP in eluted material high sensitivity is especially important. Thus, the cFMI using the Luminex system has definite advantages over a conventional ELISA including the possibility that samples can be assayed at higher dilutions. We have determined that the VIP concentrations of serum, plasma, and dried blood spot eluate specimens as measured with the cFMI assay system were similar to those measured with ELISA. Thus, the new cFMI using Luminex system may be useful for detection of VIP in human blood samples.

How common are gastrointestinal disorders in children with autism?

We could identify no report that describes the prevalence of gastrointestinal disorders in a representative group of children with a diagnosis of autism compared with appropriate controls. Thus, we found no evidence upon which to base a confident conclusion as to whether gastrointestinal symptoms are more common in children with than without autism. However, the frequency of gastrointestinal symptoms observed in population-based samples of autistic children indicate that gastrointestinal problems are not nearly as common in children with autism as reports from pediatric gastroenterology clinics suggest.