Effects of two calcium silicate cements on cell viability, angiogenic growth factor release and related gene expression in stem cells from the apical papilla.

AIM: To evaluate the effects of two types of calcium silicate cements on viability, angiogenic growth factor release, and angiogenic and inflammation-related gene expression in human stem cells from the apical papilla (SCAP). METHODOLOGY: SCAPs were grown for 7 days with either ProRoot mineral trioxide aggregate (MTA) or Biodentine (BD). Cell viability and media concentrations of vascular endothelial growth factor (VEGF/VEGFA) and angiopoietin 1 (ANGPT1) were measured. The expression of genes related to angiogenic potential and inflammatory response was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). One-way and two-way analyses of variance with multiple comparisons Tukey's test were performed (P < 0.05). RESULTS: Cells in contact with either cement were associated with increased cell viability compared with the no-treatment group at day 1 but there were no differences amongst groups at days 3 and 7. Exposure to either cement significantly increased VEGF concentrations at day 3; however, ANGPT-1 levels decreased significantly compared with the no-treatment group at day 3. Exposure to MTA and BD stimulated expression of VEGFA and FIGF/VEGFD. Furthermore, exposure to both cements significantly decreased the mRNA levels of ANGPT1 and FGF2 relative to the no-treatment group. CONCLUSIONS: Both MTA and BD stimulated the expression of angiogenic genes and release of VEGF, inducing similar expression patterns; however, they appeared to inhibit the expression of specific genes, including ANGPT1 and FGF2.

Interactions between immune system and mesenchymal stem cells in dental pulp and periapical tissues.

The recent isolation and characterization of mesenchymal stem cells (MSCs) in dental tissues constitutes a major step forward in the development of new treatment strategies. MSCs are essential for dental pulp repair and the success of regenerative endodontic procedures. It is important to understand that immune cells and cytokines can affect stem cell function, which can impact their healing potential. On the other hand, stem cells are immunoprivileged and have the ability to modulate immune and inflammatory responses, which can be utilized to improve treatments outcome. This review addresses both aspects of this interaction and suggests that any change on both sides can tip the balance in favour of either persistence of inflammation or healing. Finally, the therapeutic relevance of the interaction between MSCs and immune system relative to current treatments is discussed, and future research and treatment perspectives are suggested.

Human analogue of the morris water maze for testing subjects at risk of Alzheimer's disease.

BACKGROUND: Patients with Alzheimer's disease (AD) and amnestic mild cognitive impairment (MCI) have difficulties with spatial orientation. OBJECTIVE: To test hypothesis that spatial navigation is impaired early in MCI patients representing the presymptomatic stage of AD. METHODS: We tested patients with probable AD (n = 21), MCI, further classified according to Petersen's criteria as amnestic MCI (aMCI) single domain (n = 11), aMCI multiple domain (n = 31), or nonamnestic MCI (n = 7). The aMCI group was also stratified using cued recall according to Dubois' criteria into memory impairment of the hippocampal type (n = 10) and isolated memory retrieval impairment-nonhippocampal (n = 32) and also according to ApoE4 status into E4+ (n = 12) and E4- (n = 30). These patients and controls (n = 28) were tested in the human variant of the Morris water maze. Depending on the subtest, the subjects could use the egocentric or allocentric (hippocampus-dependent) navigation. RESULTS: The AD and aMCI multiple domain groups were impaired in all subtests. The aMCI single domain group was impaired in allocentric subtests. The hippocampal MCI group performed poorer than the nonhippocampal MCI group and similarly to the AD group. The ApoE4+ group was as bad as the AD group when compared with the E4- group. CONCLUSION: aMCI subjects represent a very heterogeneous population, and spatial memory or cued recall examination can add more value to aMCI classification. ApoE4+ patients are more impaired than ApoE4- patients.

Lipoxin A4 Attenuates the Inflammatory Response in Stem Cells of the Apical Papilla via ALX/FPR2.

Similar to the onset phase of inflammation, its resolution is a process that unfolds in a manner that is coordinated and regulated by a panel of mediators. Lipoxin A4 (LXA4) has been implicated as an anti-inflammatory, pro-resolving mediator. We hypothesized that LXA4 attenuates or prevents an inflammatory response via the immunosuppressive activity of Stem Cells of the Apical Papilla (SCAP). Here, we report for the first time in vitro that in a SCAP population, lipoxin receptor ALX/FPR2 was constitutively expressed and upregulated after stimulation with lipopolysaccharide and/or TNF-α. Moreover, LXA4 significantly enhanced proliferation, migration, and wound healing capacity of SCAP through the activation of its receptor, ALX/FPR2. Cytokine, chemokine and growth factor secretion by SCAP was inhibited in a dose dependent manner by LXA4. Finally, LXA4 enhanced immunomodulatory properties of SCAP towards Peripheral Blood Mononuclear Cells. These findings provide the first evidence that the LXA4-ALX/FPR2 axis in SCAP regulates inflammatory mediators and enhances immunomodulatory properties. Such features of SCAP may also support the role of these cells in the resolution phase of inflammation and suggest a novel molecular target for ALX/FPR2 receptor to enhance a stem cell-mediated pro-resolving pathway.

Clinical Effects of Tramiprosate in APOE4/4 Homozygous Patients with Mild Alzheimer's Disease Suggest Disease Modification Potential.

BACKGROUND: Alzheimer's Disease (AD) patients homozygous for the APOE4 allele (APOE4/4) have a distinct clinical and biological phenotype with high levels of beta amyloid (Aß) pathology and toxic Aß oligomers. Tramiprosate, an oral agent that inhibits Aß monomer aggregation into toxic oligomers, was evaluated in two Phase 3 Mild to Moderate AD studies which did not show efficacy in the overall population. Re-analyses of these trials showed the most consistent clinical benefits in APOE4/4 patients. We analyzed efficacy in the APOE4/4 patients with Mild disease. OBJECTIVES: To determine the optimal stage of AD for future trials in APOE4/4 homozygotes. DESIGN: Two randomized, double-blind, placebo-controlled parallel-arm multi-center studies of 78-weeks duration. SETTING: Academic Alzheimer's disease centers, community-based memory clinics, and neuropsychiatric research sites. PARTICIPANTS: Participants included 2,025 AD patients with MMSE 16-26. Approximately 13-15% had APOE4/4 genotype (N= 147 and 110 per study), mean age 71.1 years, 56% females. Almost all were on stable symptomatic drugs. INTERVENTION: Randomized subjects received oral placebo, 100mg BID, or 150mg BID of tramiprosate. MEASUREMENTS: Co-primary outcomes were change from baseline in the ADAS-cog11 and CDR-SB. Disability assessment for dementia (DAD) was a secondary outcome. RESULTS: In APOE4/4 homozygotes receiving 150mg BID tramiprosate, efficacy in the traditional Mild AD patients (MMSE 20-26) was higher than the overall group (MMSE 16-26) and efficacy in the Mild patients (MMSE 22-26) was highest. Tramiprosate benefits compared to placebo on ADAS-cog, CDR-SB, and DAD were 125%, 81% and 71%, respectively (p<0.02). The Mild subgroup (MMSE 22-26) showed cognitive stabilization with no decline over 78 weeks, both ADAS-cog and DAD effects increased over time. Tramiprosate safety in APOE4/4 patients was favorable. Most common adverse events were nausea, vomiting, depression and decreased weight. CONCLUSIONS: The Mild subgroup of APOE4/4 AD patients (MMSE 22-26) showed larger benefits on the high dose of tramiprosate than the overall Mild and Moderate group. Consistent with its preclinical effects on Aß oligomers, tramiprosate seemed to stabilize cognitive performance, supporting its disease modification potential. Confirmatory studies using ALZ-801, an improved pro-drug formulation of tramiprosate, will target APOE4/4 patients with Mild AD.

Clinical Benefits of Tramiprosate in Alzheimer's Disease Are Associated with Higher Number of APOE4 Alleles: The "APOE4 Gene-Dose Effect".

BACKGROUND: Tramiprosate is an oral amyloid anti-aggregation agent that reduces amyloid oligomer toxicity in preclinical studies and was evaluated in two 78-week trials in North America and Western Europe that enrolled 2,025 patients with Mild to Moderate Alzheimer's Disease. The completed North American study did not achieve its efficacy objectives, but a pre-specified subgroup analysis suggested potential efficacy in apolipoprotein E4 (APOE4) carriers. To further explore this observation, we analyzed tramiprosate Phase 3 clinical data based on the number of APOE4 alleles. OBJECTIVES: To analyze tramiprosate efficacy, safety, and occurrence of vasogenic edema in the three APOE4 subgroups: homozygous, heterozygous and non-carriers. DESIGN: Randomized, double-blind, placebo-controlled parallel-arm multi-center studies. SETTING: Academic Alzheimer's disease and dementia centers, community-based dementia and memory clinics, and neuropsychiatric clinical research sites. PARTICIPANTS: Subjects included 2,025 patients, 50 years of age or older, with approximately 60% having APOE4 carrier status (10-15% homozygotes and 45-50% heterozygotes), and mild to moderate disease. All subjects were on stable symptomatic drugs. INTERVENTION: Randomized subjects received placebo, 100 mg BID, or 150 mg BID of tramiprosate. MEASUREMENTS: Co-primary outcomes in both studies were change from baseline in the ADAS-cog11 and CDR-SB assessment scales. RESULTS: Highest efficacy was observed in APOE4/4 homozygotes receiving 150 mg BID of tramiprosate, showing statistically significant effects on ADAS-cog and positive trends on CDR-SB (respectively, 40-66% and 25-45% benefit compared to placebo). APOE4 heterozygotes showed intermediate efficacy, and non-carriers showed no benefit. In 426 patients with MRI scans, no cases of treatment-emergent vasogenic edema were observed. In the three subgroups, the most common adverse events were nausea, vomiting, and decreased weight. CONCLUSIONS: The "APOE4 Gene-Dose effect" is likely explained by the high prevalence of amyloid pathology in symptomatic APOE4 carriers. In APOE4/4 Alzheimer's disease patients, the high dose of tramiprosate showed favorable safety and clinically meaningful efficacy in addition to standard of care.

Truncated apolipoprotein E (ApoE) causes increased intracellular calcium and may mediate ApoE neurotoxicity.

Apolipoprotein E (apoE)-related synthetic peptides, the 22 kDa N-terminal thrombin-cleavage fragment of apoE (truncated apoE), and full-length apoE have all been shown to exhibit neurotoxic activity under certain culture conditions. In the present study, protease inhibitors reduced the neurotoxicity and proteolysis of full-length apoE but did not block the toxicity of truncated apoE or a synthetic apoE peptide, suggesting that fragments of apoE may account for its toxicity. Additional experiments demonstrated that both truncated apoE and the apoE peptide elicit an increase in intracellular calcium levels and subsequent death of embryonic rat hippocampal neurons in culture. Similar effects on calcium were found when the apoE peptide was applied to chick sympathetic neurons. The rise in intracellular calcium and the hippocampal cell death caused by the apoE peptide were significantly reduced by receptor-associated protein, removal of extracellular calcium, or administration of the specific NMDA glutamate receptor antagonist MK-801. These results suggest that apoE may be a source of both neurotoxicity and calcium influx that involves cell surface receptors. Such findings strengthen the hypothesis that apoE plays a direct role in the pathology of Alzheimer's disease.

Single K+ currents during differentiation of embryonic muscle cells in vitro.

After 3-7 days in culture, chicken myotubes possess five types of K+ channel: two high-conductance channels of 195 and 105 pS which are sensitive to tetraethylammonium (TEA), an ATP-sensitive channel of 64 pS and two low-conductance channels of 40 and 15 pS which are insensitive to TEA and ATP. The same population of channels is to be found in EGTA-treated muscle cells with blocked fusion and, with the exception of the ATP-sensitive channel, also in 1-day-old myoblasts. There are differences between myoblasts and myotubes in the percentage of incidence of individual channel types. High-conductance K+ channels are most frequently to be observed in myotubes, but they are rare in myoblasts and EGTA-treated cells where low-conductance K+ channels predominate.

Poly(2-hydroxyethyl methacrylate)--collagen composites which promote muscle cell differentiation in vitro.

A new simple method has been developed which allows the mixing of poly(2-hydroxyethyl methacrylate)--polyHEMA--and fibrillar collagen in any desired ratio. PolyHEMA alone was shown to be an unsuitable cultivation substrate for primary cultures of chicken embryonic skeletal muscle cells. Composites containing polyHEMA and 50% (w/w) or more collagen supported myogenesis. Such layers, firmly adhered to the bottom of plastic Petri dishes, were mechanically stable and biologically active, thus favourably combining properties of both the original materials. It is suggested that polyHEMA-collagen composite layers may be used for cultivation of differentiating cells in vitro.

A thrombin cleavage fragment of apolipoprotein E exhibits isoform-specific neurotoxicity.

A 22 kDa fragment of apoE containing a putative cytotoxi domain was identified in postmortem human brain tissue and fresh CSF. This fragment is apparently equivalent to the major apoE thrombin cleavage product. In vitro toxicity assays demonstrate that the corresponding fragment derived from recombinantly expressed human apoE is toxic to primary neurons in culture and that the E4-derived fragment is significantly more toxic than the fragment derived from the E3 isoform. These results suggest that proteolytic fragments of apoE may play a direct role in the pathology associated with AD and other diseases in which apoE has been implicated.

Sympathetic neurite outgrowth is greater on plaque-poor vs. plaque-rich regions of Alzheimer's disease cryostat sections.

Senile plaques are a characteristic histopathological feature of Alzheimer's disease (AD) and are associated with altered neuritic morphology. Numerous individual plaque components, most notably beta-amyloid, have been studied for their possible effects on neurite outgrowth in culture. However, the effect of senile plaques on neuronal morphology and function is difficult to assess. In the present study, the effect of senile plaques on neurite outgrowth was studied by culturing embryonic chick sympathetic neuronal explants on Alzheimer's tissue sections. Explants were cultured for 3 days on amygdala tissue sections from AD as well as non-AD patients in serum-free medium. Neurite outgrowth on plaque-rich regions was compared with outgrowth on plaque-poor regions of the same tissue section, and with outgrowth on non-AD tissue, through colocalization of the living explants and the underlying plaques. Explants growing on plaque-rich regions showed significantly less neurite outgrowth compared with those on plaque-poor regions in the same section or on control brain tissue. These results suggest that plaques are poor substrates for neurite outgrowth as compared with non-plaque areas of the same tissue sections, and support the hypothesis that components of the senile plaques may inhibit neurite outgrowth.

Flexor tendon repair using a stainless steel internal anchor. Biomechanical study on human cadaver tendons.

We have developed a stainless steel internal tendon anchor that is used to strengthen a tendon repair. This study tested its use in vitro to produce a repair that can withstand the tensile strength demands of early active flexion. Fresh human cadaver flexor digitorum profundus tendons were harvested, divided, and then repaired using four different techniques: Kessler, Becker or Savage stitches, or the internal tendon anchor. The internal splint repairs demonstrated a 99-270% increase in mean maximal linear tensile strength and a 49-240% increase in mean ultimate tensile strength over the other repairs. It is hoped that this newly developed internal anchor will provide a repair that will be strong enough to allow immediate active range of motion.

Cultivation conditions influencing de novo development of nerve-muscle junctions in vitro.

A variety of cultivation conditions was tested with respect to the formation of nerve-muscle junctions in vitro. The long-term maintenance and survival of innervated muscle fibres was achieved under these conditions: The myoblasts from 11 to 12-day-old chicken embryos were added to a section of the spinal cord with meninges, which was taken from 7 to 9-day-old chicken embryos and cultivated three days in advance in Maximow's assemblies. The myoblasts were prepared from pectoral or leg muscles. The spinal cord sections of the cervical, throacic and lumbar levels were equally successful in inducing the development of nerve-muscle junctions.

Neurotoxicity of the 22 kDa thrombin-cleavage fragment of apolipoprotein E and related synthetic peptides is receptor-mediated.

Potent neurotoxicity is associated with both apolipoprotein E (apoE)-related synthetic peptides and the 22 kDa N-terminal thrombin-cleavage fragment of apoE. Furthermore, the E4 isoform of the 22 kDa fragment is significantly more toxic than the same fragment derived from the E3 isoform, suggesting the possibility of a direct role of apoE-associated neurotoxicity in the pathophysiology of Alzheimer's disease. In the present study, the potential role of cell surface receptors in mediating neurotoxicity was assessed by using a variety of agents that should block the heparin-binding and receptor-binding activity of apoE. Effective inhibitors of neurotoxicity of both the apoE peptides and the apoE fragment include heparin, heparan sulfate, sodium chlorate and heparinase, the low-density lipoprotein (LDL) receptor-related protein receptor-associated protein, and a polyclonal anti-LDL receptor-related protein antibody. These results suggest that the neurotoxicity of the 22 kDa thrombin cleavage fragment of apoE and related peptides is receptor-mediated, and that the most likely candidate receptor is a heparan sulfate proteoglycan-LDL receptor-related protein complex.

Neurite degeneration elicited by apolipoprotein E peptides.

Apolipoprotein E (apoE) has been localized to the neurofibrillary tangles and beta-amyloid-containing plaques found in the cortex of patients with Alzheimer's disease (AD) (14, 28), suggesting that apoE may play a role in this disorder. Recently, synthetic peptides containing a sequence within apoE (amino acids 141-155) were found to be cytotoxic to T lymphocytes in culture. In the present study, tandem presentation of the apoE sequence E141-155, as well as longer monomeric peptides that include this domain, was found to cause extensive and specific degeneration of neurites from embryonic chick sympathetic ganglia in vitro. These results, together with the observation of strong beta A4/apoE binding in vitro and the disproportionate occurrence of the epsilon 4 allele in both familial and sporadic AD patients, suggest that peptide sequences associated with apoE may contribute directly to neurodegenerative processes.

Effect of exercise on muscle fibre composition and enzyme activities of skeletal muscles in young rats.

Young Wistar rats underwent dynamic (D) or static (S) exercise from the 5th to 35th day after birth. Histochemical and biochemical analysis were performed in the extensor digitorum longus (EDL) and the soleus muscle (SOL). Lactate dehydrogenase (LDH) (regulating anaerobic metabolism) and citrate synthase (CS) and hydroxyacyl-CoA dehydrogenase (HAD) (both regulating aerobic metabolism) activities were determined spectrophotometrically. An increase of the fast oxidative-glycolytic (FOG) muscle fibres was found in the slow SOL muscle in both trained groups, i.e. by 10% in group D and by 7% in group S in comparison with the C group. The EDL muscle fibre distribution did not differ from those of control animals in respect to the slow oxidative (SO) fibre type. A higher percentage of FOG fibres by 19% was found in group D contrary to a decreased number of the fast glycolytic (FG) muscle fibres in this trained group. The greatest increase of CS (EDL 185%, SOL 176%) and HAD (EDL 83%, SOL 178%) activities were found in group D as compared with control group (C). Only small differences were observed in LDH activity. The values of characteristic enzyme activity ratios show that dynamic training resulted in an elevation of oxidative capacity of skeletal muscle, while the static load led preferentially along the glycolytic pathway. It may be concluded that an adaptive response to the training load during early postnatal development is different due to the type of exercise (dynamic or static) and/or the type of skeletal muscle (fast or slow).