Quinolinic acid in the cerebrospinal fluid of children after traumatic brain injury

OBJECTIVE: To measure quinolinic acid, a macrophage-derived neurotoxin, in the cerebrospinal fluid (CSF) of children after traumatic brain injury (TBI) and to correlate CSF quinolinic acid concentrations to clinically important variables. DESIGN: A prospective, observational study. SETTING: The pediatric intensive care unit in Children's Hospital of Pittsburgh, a tertiary care, university-based children's hospital. PATIENTS: Seventeen critically ill children following severe TBI (Glasgow Coma Scale score <8) whose care required the placement of an intraventricular catheter for continuous drainage of CSF. Interventions: None. MEASUREMENTS AND MAIN RESULTS: Patients ranged in age from 2 mos to 16 yrs (mean 6.0 yrs). CSF was collected immediately on placement of the ventricular catheter and daily thereafter. Quinolinic acid concentration was measured by gas chromatography/mass spectroscopy in 69 samples (4.0 +/- 0.4 [SEM] samples per patient). CSF quinolinic acid concentration progressively increased after injury (p = .034, multivariate analysis) and was increased in nonsurvivors vs. survivors (p = .002, multivariate analysis). CSF quinolinic acid concentration was not associated with age. Although overall CSF quinolinic acid concentration was not associated with shaken injury (p = .16, multivariate analysis), infants suffering with shaken infant syndrome had increased admission CSF quinolinic acid concentrations compared with children with accidental mechanisms of injury (p = .027, Mann-Whitney Rank Sum test). CONCLUSIONS: A large and progressive increase in the macrophage-derived neurotoxin quinolinic acid is seen following severe TBI in children. The increase is strongly associated with increased mortality. Increased CSF quinolinic acid concentration on admission in children with shaken infant syndrome could reflect a delay in presentation to medical attention or age-related differences in quinolinic acid production. These findings raise the possibility that quinolinic acid may play a role in secondary injury after TBI in children and suggest an interaction between inflammatory and excitotoxic mechanisms of injury following TBI.     

A Highly Specific Holin-Mediated Mechanism Facilitates the Secretion of Lethal Toxin TcsL in Paeniclostridium sordellii

Protein secretion is generally mediated by a series of distinct pathways in bacteria. Recently, evidence of a novel bacterial secretion pathway involving a bacteriophage-related protein has emerged. TcdE, a holin-like protein encoded by toxigenic isolates of Clostridioides difficile, mediates the release of the large clostridial glucosylating toxins (LCGTs), TcdA and TcdB, and TpeL from C. perfringens uses another holin-like protein, TpeE, for its secretion; however, it is not yet known if TcdE or TpeE secretion is specific to these proteins. It is also unknown if other members of the LCGT-producing clostridia, including Paeniclostridium sordellii (previously Clostridium sordellii), use a similar toxin-release mechanism. Here, we confirm that each of the LCGT-producing clostridia encode functional holin-like proteins in close proximity to the toxin genes. To characterise the respective roles of these holin-like proteins in the release of the LCGTs, P. sordellii and its lethal toxin, TcsL, were used as a model. Construction and analysis of mutants of the P. sordellii tcsE (holin-like) gene demonstrated that TcsE plays a significant role in TcsL release. Proteomic analysis of the secretome from the tcsE mutant confirmed that TcsE is required for efficient TcsL secretion. Unexpectedly, comparative sample analysis showed that TcsL was the only protein significantly altered in its release, suggesting that this holin-like protein has specifically evolved to function in the release of this important virulence factor. This specificity has, to our knowledge, not been previously shown and suggests that this protein may function as part of a specific mechanism for the release of all LCGTs.     

Two common mutations in the CLN2 gene underlie late infantile neuronal ceroid lipoluscinosis

Zhong N, Wisniewski KE, Hartikainen J, Ju W, Moroziewicz DN, McLendon L, Sklower Brooks S, Brown WT. Two common mutations in the CLN2 gene underlie late infantile neuronal ceroid lipofuscinosis
Late infantile neuronal ceroid lipofuscinosis (LINCL) is one of the most common pediatric neuronal degenerative disorders. A candidate gene underlying this disease, designated CLN2, was recently cloned and the gene product was characterized as a lysosomal pepstatin-insensitive carboxypeptidase (LPIC). Four mutations were identified in CLN2 from three unrelated LINCL individuals. To investigate further the mutation frequency in LINCL, we screened 16 LINCL probands for these four mutations. The previously reported intronic mutation, T523–1 G°C, was found in 56% (9/16) of the cases, of which two were homozygous and accounted for 34% (11/32) of LINCL chromosomes. The previously reported nonsense mutation, 636 C→T leading to R208stop, was found in 31% (5/16) of the cases, including one ho-mozygote and accounted for 19% (6/32) of LINCL chromosomes. Two previously described missense mutations, 1107 T°C and 1108 G→A, were not detected in any of these 16 probands. In total, the two observed mutations, T523–1 G°C and 636 C→T, accounted for 53% (17/32) of LINCL alleles. Thus, one or both mutations were seen in 11 (69%) cases and no mutation has yet been identified in five. Our finding that these two mutations are common in LINCL cases adds further evidence in support of the idea that dysfunction of LPIC underlies LINCL. Positive molecular testing can now complement clinical diagnosis of LPIC and will allow for pre-natal diagnosis for subsequent pregnancies.     

Measurement of glucose and metabolites in subcutaneous adipose tissue during hyperglycemia with microdialysis at various perfusion flow rates

BACKGROUND: Microdialysis-based glucose sensors have recently been introduced for monitoring glucose levels in diabetic patients. The flow rate by which the fluid sample is pumped through the microdialysis catheter varies in different studies. AIM: To study the effects of various flow rates on glucose and its metabolites sampled by microdialysis during an oral glucose tolerance test. MATERIAL, METHODS: Glucose, lactate, pyruvate and glycerol were measured with microdialysis in interstitial fluid of subcutaneous adipose tissue in twelve healthy young subjects before and during an oral glucose tolerance test using four different flow rates (0.3, 1, 2 and 5 microL/min) and a 30 mm dialysis membrane. RESULTS: At the basal fasting state the dialysate glucose obtained by 0.3 microL/min was equal to capillary glucose concentration. A decrease in dialysate glucose levels during the basal state was observed for higher flow rates but not for 0.3 microL/min, which indicates a depleting effect. The relative increase after OGTT was similar for capillary glucose and flow rate 0.3 microL/min but not for higher flow rates. CONCLUSION: The low microdialysis flow rate (0.3 microL/min) facilitates the capture of true interstitial glucose concentrations during glucose fluctuations. Thus this low flow rate is preferred in studies of local tissue metabolism.     

Investigation of gene–gene interactions between CD40 and CD40L in Polish multiple sclerosis patients

CD40–CD40L interaction is necessary for the activation of both humoral and cellular immune response and has been suggested to play a role in the pathogenesis of multiple sclerosis (MS). Therefore, we analyzed the combined influence of the CD40 and CD40L variants on MS susceptibility and progression on well-defined Polish population. Our investigation revealed that CT individuals in rs1883832 locus of CD40 possessed almost 1.5-fold higher risk for MS than CC individuals (OR = 1.44; 95%CI = 1.03–2.1; p = 0.032), while this risk for TT individuals was almost 2.5-fold higher (OR = 2.36; 95%CI = 1.19–4.78; p = 0.014).     

A safer vaccine for Alzheimer's disease?

Recent reports indicate that amyloid-β (Aβ) vaccine-based therapy for Alzheimer’s disease (AD) may be on the horizon. There are, however, concerns about the safety of this approach. Immunization with Aβ1–42 may not be appropriate in humans because it crosses the blood–brain barrier, can seed fibril formation, and is highly fibrillogenic. Aβ1–42 fibrils can in turn cause inflammation and neurotoxicity. This issue is of a particular concern in the elderly who often do not mount an adequate immune response to vaccines. Our findings show that vaccination with nonamyloidogenic/nontoxic Aβ derivative may be a safer therapeutic approach to impede the progression of Aβ-related histopathology in AD. Although the site of action of the anti-Aβ antibodies has been suggested to be within the brain, peripheral clearance of Aβ may have a greater role in reducing cerebral amyloid plaques in these animals and eventually in AD patients. Antibodies in general are predominantly found outside the central nervous system (CNS) and will, therefore, primarily clear systemic Aβ compared to brain Aβ. This disruption of the equilibrium between central and peripheral Aβ should then result in efflux of Aβ out of the brain, and subsequent removal of plaques. Aβ therapy can be targeted to the periphery, which may result in fewer CNS side effects, such as inflammation. Future Aβ derived vaccines should include T_h epitopes, carriers and/or lipid moieties to enhance antibody production in the elderly, the population predominantly affected by AD.     

Mucosal vaccination delays or prevents prion infection via an oral route

In recent years major outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrP(C) (prion protein cellular), to a toxic and infectious form, PrP(Sc) (prion protein scrapie). None of the prionoses currently have an effective treatment. A limited number of active immunization approaches have been shown to slightly prolong the incubation period of prion infection. Active immunization in wild-type animals is hampered by auto-tolerance to PrP and potential toxicity. Here we report that mucosal vaccination with an attenuated Salmonella vaccine strain expressing the mouse PrP, is effective at overcoming tolerance to PrP and leads to a significant delay or prevention of prion disease in mice later exposed orally to the 139A scrapie strain. This mucosal vaccine induced gut anti-PrP immunoglobulin (Ig)A and systemic anti-PrP IgG. No toxicity was evident with this vaccination approach. This promising finding suggests that mucosal vaccination may be a useful method for overcoming tolerance to PrP and preventing prion infection among animal and potentially human populations at risk.     

Synthesis and properties of (D)- and (L)-lactide stereocopolymers using the system achiral Schiff's base/aluminium methoxide as initiator

The polymerization of lactides with various D /L enantiomeric compositions using achiral Schiff's base/aluminium methoxide as initiator (SALENAlOCH₃) in dichloromethane solution at 70°C is reported. The conversion was kept below 70% in order to limit transesterification reactions. The polymers obtained after precipitation show a narrow molecular weight distribution (ratio of weight- to number-average molecular weights M _w/M _n = 1,1–1,2) and an optical rotation higher than that expected from the optical purity of the starting monomers. The examination of the thermal properties reveals that whatever their enantiomeric composition all the prepared polymers are crystalline. This unusual behavior is explained by an end-chain propagation mechanism producing stereocopolymers with long enantiomeric sequences, i.e., increased isotacticity as substantiated by an examination of the microstructure of the polymers by means of ¹³C nuclear magnetic resonance. A stereocomplex formation was observed for stereocopolymers with optical purities below 40%.     

Presenilin-1 is associated with Alzheimer's disease amyloid.

Mutations in presenilin (PS)-1 and -2, located on chromosome 14 and 1 respectively, are the major association with early-onset familial Alzheimer's disease (FAD). FAD has also been linked to mutations in the amyloid beta precursor protein (beta PP), and the presence of the apolipoprotein E4 allele is a risk factor for late-onset AD. The role of PS in FAD and in sporadic AD is unclear. We previously reported the presence of a PS-1 carboxyl-terminal epitope in neuritic plaques (Wisniewski T, Palha JA, Ghiso J, Frangione B: S182 protein in Alzheimer's disease neuritic plaques. Lancet 1995, 346:1366). In the present study, we examined a number of biochemically different cerebral and systemic amyloidoses, finding the PS-1 carboxy epitope only in association with amyloid beta (A beta) lesions. We confirm the presence of this epitope ultrastructurally in neuritic plaques. In addition, biochemical and amino acid sequence data are presented for an association of the 18-kd carboxy fragment of PS-1 with neuritic plaques with a start at residue 300. Three of the proteins with linkage to AD have now been found as components of neuritic plaques. It remains to be determined whether all of these proteins are involved in the same or different pathological pathway(s) and which of these proteins is the most important for the common, late-onset form of AD.     

Prion protein is ubiquitinated after developing protease resistance in the brains of scrapie-infected mice

Although the key event in the pathology of prion diseases is thought to be the conversion of cellular prion protein (PrP(C)) to the protease-resistant scrapie species termed PrP(Sc), the factors that contribute to neurodegeneration in scrapie-infected animals are poorly understood. One probable determinant could be when the accumulation of PrP(Sc) in infected brain overwhelms the ubiquitin-proteasome system and triggers the degenerative cascade. In the present study, it was found that in mouse brains infected with the ME7 scrapie strain, the level of ubiquitin protein conjugates increased significantly at approximately 144 days post-infection (pi) when clinical signs first become apparent. This elevation correlated with the detection of protease-resistant PrP(Sc) and a decline in two endopeptidase activities associated with proteasome function. However, ubiquitination of PrP was only detected at the terminal stage, 3 weeks after the development of clinical symptoms (approximately 165 days pi). These results suggest that ubiquitination of PrP is a late event phenomenon and this conjugation occurs after the formation of protease-resistant PrP(Sc). Whether this post-translational modification and the impairment of proteasome function are pivotal events in the pathogenesis of prion diseases remains to be determined.