The importance of personalization in high altitude protocols for hematologic and metabolic benefits in sports: A multi-dimensional N-of-1 case study.

The hematologic and metabolic benefits of high altitude exposure have been extensively studied in athletes due to their promising performance enhancing effects. However, despite the increased research and development of various high altitude protocols for achieving peak performance, the reproducibility of the results at the individual level remains sparse. To systematically address this limitation and establish a more effective method to achieve consistent results at the individual level, we conducted a multi-dimensional study of one elite endurance athlete in two Phases. In Phase 1, we applied the standard protocol of LHTH (Live-High-Train-High) using a commercially available, at-home, normobaric, high altitude simulation tent under the SHTL (Sleep-High-Train-Low) model. Then, we developed the athlete's personalized protocol for peak hematologic parameters during their off-season. This protocol determined the exact total high altitude exposure time required to achieve peak hematologic parameters, which in the case of this athlete, amounted to 45 nights with approximately 8hrs per night. In Phase 2, we replicated the Phase 1 protocol during the athlete's in-season and observed the same or even higher hematologic and metabolic benefits compared to Phase 1. During both phases, we collected thousands of multi-dimensional data points to ensure that the athlete's lifestyle and environmental factors remained stable, and to increase the likelihood that physiological changes resulted primarily from the high altitude exposure. The data trends in both Phases validated that, for this athlete, hematologic measures such as red blood cell count, hematocrit, and hemoglobin, as well as electrolyte content, body weight and gut microbiome composition improved to their personal best values after a total of approximately 15 days of high altitude exposure (45 nights with roughly 8hrs per night totaling 360hrs or 15days). These improvements did not occur after the 21 days recommended by the LHTH protocol highlighting the significance of personalization in high altitude protocols that are designed for peak performance parameters. Therefore, to maximize the benefits in hematologic and other metabolic values and thus increase muscle oxygen supply and peak aerobic capacity through high altitude exposure, each athlete may require a unique total duration of high altitude exposure tailored to their individual physiology. This duration must be determined by their specific response in hematologic peaking. Therefore, initially establishing a personalized protocol for an athlete by determining their required total duration of high altitude exposure for peak hematologic values during their off-season and applying this protocol during their in-season phase may lead to more successful and reproducible benefits compared to following a generalized protocol alone.

Mast cell activation and autism.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by varying degrees of dysfunctional communication and social interactions, repetitive and stereotypic behaviors, as well as learning and sensory deficits. Despite the impressive rise in the prevalence of autism during the last two decades, there are few if any clues for its pathogenesis, early detection or treatment. Increasing evidence indicates high brain expression of pro-inflammatory cytokines and the presence of circulating antibodies against brain proteins. A number of papers, mostly based on parental reporting on their children's health problems, suggest that ASD children may present with "allergic-like" problems in the absence of elevated serum IgE and chronic urticaria. These findings suggest non-allergic mast cell activation, probably in response to environmental and stress triggers that could contribute to inflammation. In utero inflammation can lead to preterm labor and has itself been strongly associated with adverse neurodevelopmental outcomes. Premature babies have about four times higher risk of developing ASD and are also more vulnerable to infections, while delayed development of their gut-blood-brain barriers makes exposure to potential neurotoxins likely. Perinatal mast cell activation by infectious, stress-related, environmental or allergic triggers can lead to release of pro-inflammatory and neurotoxic molecules, thus contributing to brain inflammation and ASD pathogenesis, at least in a subgroup of ASD patients. This article is part of a Special Issue entitled: Mast cells in inflammation.

Mast cells and inflammation.

Mast cells are well known for their role in allergic and anaphylactic reactions, as well as their involvement in acquired and innate immunity. Increasing evidence now implicates mast cells in inflammatory diseases where they are activated by non-allergic triggers, such as neuropeptides and cytokines, often exerting synergistic effects as in the case of IL-33 and neurotensin. Mast cells can also release pro-inflammatory mediators selectively without degranulation. In particular, IL-1 induces selective release of IL-6, while corticotropin-releasing hormone secreted under stress induces the release of vascular endothelial growth factor. Many inflammatory diseases involve mast cells in cross-talk with T cells, such as atopic dermatitis, psoriasis and multiple sclerosis, which all worsen by stress. How mast cell differential responses are regulated is still unresolved. Preliminary evidence suggests that mitochondrial function and dynamics control mast cell degranulation, but not selective release. Recent findings also indicate that mast cells have immunomodulatory properties. Understanding selective release of mediators could explain how mast cells participate in numerous diverse biologic processes, and how they exert both immunostimulatory and immunosuppressive actions. Unraveling selective mast cell secretion could also help develop unique mast cell inhibitors with novel therapeutic applications. This article is part of a Special Issue entitled: Mast cells in inflammation.

TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation.

Tuberous sclerosis complex human disease gene products TSC1 and TSC2 form a functional complex that negatively regulates target of rapamycin (TOR), an evolutionarily conserved kinase that plays a central role in cell growth and metabolism. Here, we describe a novel role of TSC1/2 in controlling stem cell maintenance. We show that in the Drosophila ovary, disruption of either the Tsc1 or Tsc2 gene in germline stem cells (GSCs) leads to precocious GSC differentiation and loss. The GSC loss can be rescued by treatment with TORC1 inhibitor rapamycin, or by eliminating S6K, a TORC1 downstream effecter, suggesting that precocious differentiation of Tsc1/2 mutant GSC is due to hyperactivation of TORC1. One well-studied mechanism for GSC maintenance is that BMP signals from the niche directly repress the expression of a differentiation-promoting gene bag of marbles (bam) in GSCs. In Tsc1/2 mutant GSCs, BMP signalling activity is downregulated, but bam expression is still repressed. Moreover, Tsc1 bam double mutant GSCs could differentiate into early cystocytes, suggesting that TSC1/2 controls GSC differentiation via both BMP-Bam-dependent and -independent pathways. Taken together, these results suggest that TSC prevents precocious GSC differentiation by inhibiting TORC1 activity and subsequently differentiation-promoting programs. As TSC1/2-TORC1 signalling is highly conserved from Drosophila to mammals, it could have a similar role in controlling stem cell behaviour in mammals, including humans.

IL-33 augments substance P-induced VEGF secretion from human mast cells and is increased in psoriatic skin.

The peptide substance P (SP) has been implicated in inflammatory conditions, such as psoriasis, where mast cells and VEGF are increased. A relationship between SP and VEGF has not been well studied, nor has any interaction with the proinflammatory cytokines, especially IL-33. Here we report that SP (0.1-10 microM) induces gene expression and secretion of VEGF from human LAD2 mast cells and human umbilical core blood-derived cultured mast cells (hCBMCs). This effect is significantly increased by coadministration of IL-33 (5-100 ng/mL) in both cell types. The effect of SP on VEGF release is inhibited by treatment with the NK-1 receptor antagonist 733,060. SP rapidly increases cytosolic calcium, and so does IL-33 to a smaller extent; the addition of IL-33 augments the calcium increase. SP-induced VEGF production involves calcium-dependent PKC isoforms, as well as the ERK and JNK MAPKs. Gene expression of IL-33 and histidine decarboxylase (HDC), an indicator of mast cell presence/activation, is significantly increased in affected and unaffected (at least 15 cm away from the lesion) psoriatic skin, as compared with normal control skin. Immunohistochemistry indicates that IL-33 is associated with endothelial cells in both the unaffected and affected sites, but is stronger and also associated with immune cells in the affected site. These results imply that functional interactions among SP, IL-33, and mast cells leading to VEGF release contribute to inflammatory conditions, such as the psoriasis, a nonallergic hyperproliferative skin inflammatory disorder with a neurogenic component.

Relationships between body composition, anthropometrics, and standard lipid panels in a normative population.

Introduction: More than one third of adults in the United States (US) meet the clinical criteria for a diagnosis of metabolic syndrome, but often diagnosis is challenging due to healthcare access, costs and discomfort with the process and invasiveness associated with a standard medical examination. Less invasive and more accessible approaches to collecting biometric data may have utility in identifying individuals at risk of diagnoses, such as metabolic syndrome or dyslipidemia diagnoses. Body composition is one such source of biometric data that can be non-invasively acquired in a home or community setting that may provide insight into an individual's propensity for a metabolic syndrome diagnosis. Here we investigate possible associations between body composition, anthropometrics and lipid panels in a normative population. Methods: Healthy participants visited the Lab100 clinic location at a hospital setting in New York City and engaged in a wellness visit led by a nurse practitioner. Blood was analyzed at point-of-care using the Abbott Piccolo Xpress portable diagnostic analyzer (Abbott Laboratories, IL, USA) and produced direct measures of total cholesterol (TC), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C), very-low density lipoprotein (VLDL-C), and triglycerides (TG). Body composition and anthropometric data were collected using two separate pieces of equipment during the same visit (Fit3D and InBody570). Regression analysis was performed to evaluate associations between all variables, after adjusting for age, sex, race, AUDIT-C total score (alcohol use), and current smoking status. Results: Data from 199 participants were included in the analysis. After adjusting for variables, percentage body fat (%BF) and visceral fat levels were significantly associated with every laboratory lipid value, while waist-to-hip ratio also showed some significant associations. The strongest associations were detected between %BF and VLDL-C cholesterol levels (t = 4.53, p = 0.0001) and Triglyceride levels (t = 4.51, p = 0.0001). Discussion: This initial, exploratory analysis shows early feasibility in using body composition and anthropometric data, that can easily be acquired in community settings, to identify people with dyslipidemia in a normative population.

Mitochondria distinguish granule-stored from de novo synthesized tumor necrosis factor secretion in human mast cells.

BACKGROUND: Mast cells are immune cells derived from hematopoietic precursors that mature in the tissue microenvironment. Mast cells are critical for allergic, immune and inflammatory processes, many of which involve tumor necrosis factor (TNF). These cells uniquely store TNF in their secretory granules. Upon stimulation, mast cells rapidly (30 min) secrete ß-hexosaminidase and granule-stored TNF through degranulation, but also increase TNF mRNA and release de novo synthesized TNF 24 h later. The regulation of these two distinct pathways is poorly understood. METHODS: Human LAD2 leukemic mast cells are stimulated by substance P. TNF secretion and gene expression were measured by ELISA and real-time PCR, and mitochondrial dynamics was observed in live cells under confocal microscopy. Cell energy consumption was measured in terms of oxygen consumption rate. RESULTS: Here, we show that granule-stored TNF is preformed, and its secretion from LAD2 mast cells stimulated by substance P (1) exhibits higher energy consumption and is inhibited by the mitochondrial ATP pump blocker oligomycin, (2) shows rapid increase in intracellular calcium levels, and (3) exhibits reversible mitochondrial translocation, from a perinuclear distribution to the cell surface, as compared to de novo synthesized TNF release induced by lipopolysaccharide. This mitochondrial translocation is confirmed using primary human umbilical cord blood-derived mast cells stimulated by an allergic trigger (IgE/streptavidin). CONCLUSION: Our findings indicate that unique mitochondrial functions distinguish granule-stored from newly synthesized TNF release from human mast cells, thus permitting the versatile involvement of mast cells in different biological processes.

Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: relevance to atopic dermatitis.

BACKGROUND: Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. OBJECTIVE: We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. METHODS: Human umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. RESULTS: Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. CONCLUSION: Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD.

A Wipe-Based Stool Collection and Preservation Kit for Microbiome Community Profiling.

While a range of methods for stool collection exist, many require complicated, self-directed protocols and stool transfer. In this study, we introduce and validate a novel, wipe-based approach to fecal sample collection and stabilization for metagenomics analysis. A total of 72 samples were collected across four different preservation types: freezing at -20°C, room temperature storage, a commercial DNA preservation kit, and a dissolvable wipe used with DESS (dimethyl sulfoxide, ethylenediaminetetraacetic acid, sodium chloride) solution. These samples were sequenced and analyzed for taxonomic abundance metrics, bacterial metabolic pathway classification, and diversity analysis. Overall, the DESS wipe results validated the use of a wipe-based capture method to collect stool samples for microbiome analysis, showing an R2 of 0.96 for species across all kingdoms, as well as exhibiting a maintenance of Shannon diversity (3.1-3.3) and species richness (151-159) compared to frozen samples. Moreover, DESS showed comparable performance to the commercially available preservation kit (R2 of 0.98), and samples consistently clustered by subject across each method. These data support that the DESS wipe method can be used for stable, room temperature collection and transport of human stool specimens.

Neuro-inflammation, blood-brain barrier, seizures and autism.

Many children with Autism Spectrum Diseases (ASD) present with seizure activity, but the pathogenesis is not understood. Recent evidence indicates that neuro-inflammation could contribute to seizures. We hypothesize that brain mast cell activation due to allergic, environmental and/or stress triggers could lead to focal disruption of the blood-brain barrier and neuro-inflammation, thus contributing to the development of seizures. Treating neuro-inflammation may be useful when anti-seizure medications are ineffective.

Mercury induces inflammatory mediator release from human mast cells.

BACKGROUND: Mercury is known to be neurotoxic, but its effects on the immune system are less well known. Mast cells are involved in allergic reactions, but also in innate and acquired immunity, as well as in inflammation. Many patients with Autism Spectrum Disorders (ASD) have "allergic" symptoms; moreover, the prevalence of ASD in patients with mastocytosis, characterized by numerous hyperactive mast cells in most tissues, is 10-fold higher than the general population suggesting mast cell involvement. We, therefore, investigated the effect of mercuric chloride (HgCl2) on human mast cell activation. METHODS: Human leukemic cultured LAD2 mast cells and normal human umbilical cord blood-derived cultured mast cells (hCBMCs) were stimulated by HgCl2 (0.1-10 microM) for either 10 min for beta-hexosaminidase release or 24 hr for measuring vascular endothelial growth factor (VEGF) and IL-6 release by ELISA. RESULTS: HgCl2 induced a 2-fold increase in beta-hexosaminidase release, and also significant VEGF release at 0.1 and 1 microM (311 +/- 32 pg/106 cells and 443 +/- 143 pg/106 cells, respectively) from LAD2 mast cells compared to control cells (227 +/- 17 pg/106 cells, n = 5, p < 0.05). Addition of HgCl2 (0.1 microM) to the proinflammatory neuropeptide substance P (SP, 0.1 microM) had synergestic action in inducing VEGF from LAD2 mast cells. HgCl2 also stimulated significant VEGF release (360 +/- 100 pg/106 cells at 1 microM, n = 5, p < 0.05) from hCBMCs compared to control cells (182 +/- 57 pg/106 cells), and IL-6 release (466 +/- 57 pg/106 cells at 0.1 microM) compared to untreated cells (13 +/- 25 pg/106 cells, n = 5, p < 0.05). Addition of HgCl2 (0.1 microM) to SP (5 microM) further increased IL-6 release. CONCLUSIONS: HgCl2 stimulates VEGF and IL-6 release from human mast cells. This phenomenon could disrupt the blood-brain-barrier and permit brain inflammation. As a result, the findings of the present study provide a biological mechanism for how low levels of mercury may contribute to ASD pathogenesis.

Neurotensin is increased in serum of young children with autistic disorder.

Autism spectrum disorders (ASD) are a group of pervasive neurodevelopmental disorders diagnosed in early childhood. They are associated with a set of "core symptoms" that include disabilities in social interaction skills, verbal and non-verbal communication, as well as repetitive and stereotypic behaviors. There is no definite pathogenetic mechanism or diagnostic tests. Many children with ASD also have "allergic-like" symptoms, but test negative implying mast cell activation by non-allergic triggers. We measured by Milliplex arrays serum levels of 3 neuropeptides that could stimulate mast cells in children with autistic disorder (n = 19; 16 males and 3 females; mean age 3.0 ± 0.4 years) and healthy, unrelated controls (n = 16; 13 males and 3 females; mean age 3 ± 1.2 years). Only neurotensin (NT) was significantly increased from 60.5 ± 6.0 pg/ml in controls to 105.6 ± 12.4 pg/ml in autistic disorder (p = 0.004). There was no statistically significant difference in the serum levels of ß-endorphin or substance P (SP). NT could stimulate immune cells, especially mast cells, and/or have direct effects on brain inflammation and ASD.

Mitochondrial DNA and anti-mitochondrial antibodies in serum of autistic children.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by difficulties in communication, cognitive and learning deficits, as well as stereotypic behaviors. For the majority of cases there are no reliable biomarkers or distinct pathogenesis. However, increasing evidence indicates ASD may be associated with some immune dysregulation, and may have a neuroimmune component. We recently showed that the peptide neurotensin (NT) is increased in autistic children. We now show that NT induces release of extracellular mitochondrial DNA (mtDNA) that could act as "autoimmune" trigger. We further show that serum from young autistic patients contains mtDNA (n = 20; cytochrome B, p = 0.0002 and 7S, p = 0.006), and anti-mitochondrial antibody Type 2 (n = 14; p = 0.001) as compared to normally developing, unrelated controls (n = 12). Extracellular blood mtDNA and other components may characterize an autistic endophenotype and may contribute to its pathogenesis by activating autoimmune responses.

Rupatadine inhibits proinflammatory mediator secretion from human mast cells triggered by different stimuli.

BACKGROUND: Mast cells are involved in allergy and inflammation by secreting multiple mediators including histamine, cytokines and platelet-activating factor. Certain histamine 1 receptor antagonists have been reported to inhibit histamine secretion, but the effect on cytokine release from human mast cells triggered by allergic and other stimuli is not well known. We investigated the ability of rupatadine, a potent histamine 1 receptor antagonist that also blocks platelet-activating factor actions, to also inhibit mast cell mediator release. METHODS: Rupatadine (1-50 microM) was used before stimulation by: (1) interleukin (IL)-1 to induce IL-6 from human leukemic mast cells (HMC-1 cells), (2) substance P for histamine, IL-8 and vascular endothelial growth factor release from LAD2 cells, and (3) IgE/anti-IgE for cytokine release from human cord blood-derived cultured mast cells. Mediators were measured in the supernatant fluid by ELISA or by Milliplex microbead arrays. RESULTS: Rupatadine (10-50 microM) inhibited IL-6 release (80% at 50 microM) from HMC-1 cells, whether added 10 min or 24 h prior to stimulation. Rupatadine (10-50 microM for 10 min) inhibited IL-8 (80%), vascular endothelial growth factor (73%) and histamine (88%) release from LAD2 cells, as well as IL-6, IL-8, IL-10, IL-13 and tumor necrosis factor release from human cord blood-derived cultured mast cells. CONCLUSION: Rupatadine can inhibit histamine and cytokine secretion from human mast cells in response to allergic, immune and neuropeptide triggers. These actions endow rupatadine with unique properties in treating allergic inflammation, especially perennial rhinitis and idiopathic urticaria.

Improved gastrointestinal health for irritable bowel syndrome with metagenome-guided interventions.

Irritable bowel syndrome (IBS) is the most prevalent functional gastrointestinal disorder worldwide, and the most common reason for referral to gastroenterology clinics. However, the pathophysiology is still not fully understood and consequently current management guidelines are very symptom-specific, leading to mixed results. Here we present a study of 88 individuals with IBS who had baseline sequencing of their gut microbiome (stool samples), received targeted interventions that included dietary, supplement, prebiotic/probiotic, and lifestyle recommendations for a 30-day period, and a follow-up sequencing of their gut microbiome. The study's objectives were to demonstrate unique metagenomic signatures across the IBS phenotypes and to validate whether metagenomic-guided interventions could lead to improvement of symptom scores in individuals with IBS. Enrolled subjects also completed a baseline and post-intervention questionnaire that assessed their symptom scores. The average symptom score of an individual with IBS at baseline was 160 and at the endpoint of the study the average symptom score of the cohort was 100.9. The mixed IBS subtype showed the most significant reduction in symptom scores across the different subtypes (average decrease by 102 points, P = 0.005). The metagenomics analysis reveals shifts in the microbiome post-intervention that have been cross-validated with the literature as being associated with improvement of IBS symptoms. Given the complex nature of IBS, further studies with larger sample sizes, more targeted analyses, and a broader population cohort are needed to explore these results further.

Finding value in the U.S. Food and Drug Administration's Fast Track program.

The U.S. Food and Drug Administration's (FDA) Fast Track program, created in 1997, was designed to facilitate the development and expedite the review of drugs and biologics intended to treat serious or life-threatening conditions, and that demonstrate the potential to address unmet medical needs. Although the intent is laudable, the significance of designations and effectiveness of the program have recently come into question. Tufts Center for the Study of Drug Development has collected data on fast track candidates since 1998. We analyzed the current dataset of 344 fast track candidates granted nearly 400 designations, representing approximately 70% of the fast track designations granted by FDA, to address questions regarding common metrics. We found that fast track candidates were widely diverse in characteristics and development histories. The complexity and limitations of the data introduced biases in metrics such as clinical phase lengths and phase transition probabilities, although these could be determined for subsets of the candidates. Our results suggest that evaluation of the Fast Track program requires a nuanced approach, and estimates of the program's value should include assessment of the resulting marketed products.

Reversal effect of Ganoderma lucidum polysaccharide on multidrug resistance in K562/ADM cell line.

AIM: To investigate the reversal effect of Ganoderma lucidum polysaccharides (Gl-PS) on multidrug resistance (MDR) in the adriamycin (ADM)-resistant leukemic cell line K562/ADM. METHODS: Cytotoxicity was assayed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide method; the ADM concentration in cells was determined by flow cytometry and confocal laser scanning microscopy techniques; the expression of P-glycoprotein was assayed by flow cytometry; and the mRNA expression levels of MDR-1 and MDR-associated protein (MRP)1 were determined by RT-PCR. RESULTS: Gl-PS reversed MDR in K562/ADM cells. Gl-PS obviously reversed the resistance of K562/ADM to doxorubicin. The reversing factors of Gl-PS at 10 and 20 mg/L were 6.46 and 6.80, respectively. MDR-1 and MRP1 transcription were downregulated by 10 and 50 mg/L Gl-PS. CONCLUSION: Gl-PS can reverse the MDR by downregulating the expression of MDR-1 and MRP1 in K562/ADM cells.

Nanotube Formation: A Rapid Form of "Alarm Signaling"?

PURPOSE: Tunneling nanotubes (TNTs) are extremely thin (50-200 nm), actin-containing cell surface protrusions up to a few microns in length that can develop rapidly and connect various cell types. Mast cells (MCs) are unique immunomodulatory cells that are found perivascularly in all tissues. MCs communicate with many other cell types through the release of inflammatory, neurosensitizing, and vasoactive molecules, through which they are involved in the pathogenesis of many inflammatory diseases. We, therefore, investigated the possibility that MCs may form TNTs and communicate among themselves and with glioblastoma cells. METHODS: Laboratory Allergic Diseases (LAD)-2 human MCs were cultured in medium supplemented with 100 U/mL penicillin/streptomycin and 100 ng/mL recombinant human stem cell factor. They were incubated with 20 nmol/L deep red probe for 20 minutes and 50 nmol/L green probe for 30 minutes. Human glioblastoma cells were incubated with 20 nmol/L deep red probe only, moved to glass-bottom culture dishes, and observed using a substance P 2 confocal microscope. LAD2 MCs were stimulated with 2 µmol/L of the peptide substance P for 30 minutes at 37ºC. Confocal digital images were processed. FINDINGS: MCs can rapidly (within 5 minutes) form TNTs, which appear to transport mitochondrial and secretory granule particles among themselves and with cocultured glioblastoma cells. IMPLICATIONS: MCs are now accepted as having an important role in many diseases with an inflammatory component. TNTs provide a rapid and direct way for MCs to "alarm" other cell types with specificity not present when mediators are secreted into the tissue microenvironment. The identification of TNTs and their cargo could be important in the diagnosis and possible treatment of many inflammatory diseases.