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Ulcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory intestinal diseases that show a perplexing heterogeneity in manifestations and response to treatment. The molecular basis for this heterogeneity remains uncharacterized. We applied single-cell RNA sequencing and CosMx™ Spatial Molecular Imaging to human colon and found the highest diversity in cellular composition in the myeloid compartment of UC and CD patients. Besides resident macrophage subsets (M0 and M2), patients showed a variety of activated macrophages including classical (M1 CXCL5 and M1 ACOD1) and new inflammation-dependent alternative (IDA) macrophages. In addition, we captured intestinal neutrophils in three transcriptional states. Subepithelial IDA macrophages expressedNRG1, which promotes epithelial differentiation. In contrast,NRG1lowIDA macrophages were expanded within the submucosa and in granulomas, in proximity to abundant inflammatory fibroblasts, which we suggest may promote macrophage activation. We conclude that macrophages sense and respond to unique tissue microenvironments, potentially contributing to patient-to-patient heterogeneity.

A subset of individuals who recover from coronavirus disease 2019 (COVID-19) develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal tissue samples. The mouse-adapted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute to clinical recovery phases. At 15 to 120 days post-virus clearance, pulmonary histologic findings included subpleural lesions composed of collagen, proliferative fibroblasts, and chronic inflammation, including tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal up-regulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC., After recovery from acute SARS-CoV-2 infection, mice exhibit chronic lung disease similar to some humans, allowing for testing of therapeutics.

The first prospective noninterventional registry study (RESPOND) evaluated the clinical effectiveness of a native type I collagen matrix plus polyhexamethylene biguanide antimicrobial barrier (PCMP) in various nonhealing wounds. This product is intended for the management of partial- and full-thickness wounds and acts as an effective barrier to reduce microbes penetrating through the dressing. The RESPOND study demonstrated that PCMP has clinically meaningful benefits in managing a variety of wounds.The authors describe the effects of PCMP in the subgroup of patients with pressure injuries (PIs) from the RESPOND registry.The prospective, noninterventional study was designed to collect information regarding the use of PCMP in a real-world situation. Eligibility included male and female patients aged 18 years and older with target wounds (partial- or full-thickness) suitable for the use of PCMP. Enrolled patients were followed approximately weekly for up to 32 weeks. All wounds and the subgroups were analyzed to determine the frequency and median time to wound closure using Kaplan-Meier methods.The patients with PIs were older adults with a mean age of 69 years and a mean BMI of 27 kg/m2. At baseline, the mean measured wound length was 3 cm, the mean depth was 8.0 mm, the mean volume was 12.6 cm3, and the mean area was 10.5 cm2. Complete wound closures were evident in 5% of patients (n = 2) at week 4, and were achieved in 39% of patients (n = 18) by week 16, in 49% (n = 22) by week 24, and in 62% (n = 28) by week 32. The median time to wound closure was 32 weeks. For all 45 PIs managed with PCMP, the incidence of achieving greater than 60% reduction in baseline area and depth was 78% (n = 35) and 64% (n = 29), respectively, with approximately 82% (n = 37) of wounds showing a reduction in volume greater than 75%.It appears that PCMP is a useful adjunct in managing chronic deep wounds such as PIs.

Background: Aging is considered the most important risk factor for Alzheimer’s disease (AD). Recent research supports the theory that immunotherapy targeting the “oligomeric” forms of amyloid-β (Aβ) may halt the progression of AD. However, previous clinical trial of the vaccine against Aβ, called AN1792, was suspended due to cases of meningoencephalitis in patients. Objective: To develop a peptide sensitized dendritic cells (DCs) vaccine that would target oligomer Aβ and prevent an autoimmune response. Methods: Double transgenic APPswe/PS1ΔE9 (Tg) and C57BL/6J control mice were used in this study. Cytokine expression profile detection, characterization of antisera, brain GSK-3β, LC3 expression, and spatial working memory testing before and post-vaccination were obtained. Results: Epitope prediction indicated that E22W42 could generate 13 new T cell epitopes which can strengthen immunity in aged subjects and silence several T cell epitopes of the wild type Aβ. The silenced T cell epitope could help avoid the autoimmune response that was seen in some patients of the AN-1792 vaccine. The E22W42 not only helped sensitize bone marrow-derived DCs for the development of an oligomeric Aβ-specific antibody, but also delayed memory impairment in the APP/PS1 mouse model. Most importantly, this E22W42 peptide will not alter the DC’s natural immunomodulatory properties. Conclusion: The E22W42 vaccine is possibly safer for patients with impaired immune systems. Since there is increasing evidence that oligomeric form of Aβ are the toxic species to neurons, the E22W42 antibody’s specificity for these “oligomeric” Aβ species could provide the opportunity to produce some clinical benefits in AD subjects.

COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

BackgroundAlthough cancer immunotherapies can effectively restore T cell-mediated immunity leading to sustained clinical responses, these responses are unpredictable partly due to highly heterogeneous phenotypes of tumor-infiltrating lymphocytes (TILs) between patients. Thus, understanding such TILs and their roles in the context of tumor microenvironments (TME) may lead to developing better immunotherapy solutions. The spatial molecular imager (SMI) is a novel spatial transcriptomics platform that allows spatially resolved high-dimensional cellular phenotyping for comprehensive TIL profiling. SMI uses fluorescent molecular barcodes to enable in-situ measurement of biological targets on an intact tissue sample. Here, we characterize comprehensive TIL phenotypes and visualize landscape of TILs directly on intact formalin-fixed paraffin-embedded (FFPE) tissues using a 1000+-plex RNA panel.MethodsTo build multi-omics TIL profiling data sets for renal cell carcinoma (RCC) tissues, we employed scRNA-seq, mass cytometry (CyTOF) and SMI. Peripheral blood mononuclear cells and dissociated cells from matched RCC tumor and adjacent normal tissues were analyzed by CyTOF and single-cell sequencing. Then, SMI profiling of matching FFPE tissues was used to visualize TILs in the context of the TME and to understand relationships between high-dimensional cellular heterogeneity and the spatial organization of cells within a tumor tissue.ResultsCyTOF and scRNA-seq analysis of dissociated cells was used to determine the gene expression profiles of numerous cellular subsets. TCR sequencing was also used to assess the extent of clonal expansion and clonotypic relationships between blood and tumor. Consistent with our previous reports, T cell populations could be segregated based on markers associated with chronic T cell receptor signaling and many T cells with an exhausted phenotype were clonally expanded in the tumor but not the blood. In contrast, T cell clonotypes with bystander phenotypes in the tumor were readily detected as expanded clones in the blood, supporting notion that not all tumor-infiltrating T cells are specific for tumor antigens. SMI analysis of matched tumor tissue was used to accurately quantify the densities and to determine the spatial organization of all T cell subsets. In addition, computational methods were used to describe distinct cellular niches within tumors with accurately defined cellular compositions.ConclusionsHigh dimensional cellular profiling highlights the abundance of bystander T cell infiltration of RCC tumors. Comprehensive spatial profiling by SMI provides spatial context to the highly diverse immune cell composition of tumor infiltrates.Ethics ApprovalFully anonymous human material was obtained from Northwest Biotrust and given IRB designation of non-human subjects research.

Aging is considered the most important risk factor for Alzheimer's disease (AD). Recent research supports the theory that immunotherapy targeting the "oligomeric" forms of amyloid-β (Aβ) may halt the progression of AD. However, previous clinical trial of the vaccine against Aβ, called AN1792, was suspended due to cases of meningoencephalitis in patients.To develop a peptide sensitized dendritic cells (DCs) vaccine that would target oligomer Aβ and prevent an autoimmune response.Double transgenic APPswe/PS1ΔE9 (Tg) and C57BL/6J control mice were used in this study. Cytokine expression profile detection, characterization of antisera, brain GSK-3β, LC3 expression, and spatial working memory testing before and post-vaccination were obtained.Epitope prediction indicated that E22W42 could generate 13 new T cell epitopes which can strengthen immunity in aged subjects and silence several T cell epitopes of the wild type Aβ. The silenced T cell epitope could help avoid the autoimmune response that was seen in some patients of the AN-1792 vaccine. The E22W42 not only helped sensitize bone marrow-derived DCs for the development of an oligomeric Aβ-specific antibody, but also delayed memory impairment in the APP/PS1 mouse model. Most importantly, this E22W42 peptide will not alter the DC's natural immunomodulatory properties.The E22W42 vaccine is possibly safer for patients with impaired immune systems. Since there is increasing evidence that oligomeric form of Aβ are the toxic species to neurons, the E22W42 antibody's specificity for these "oligomeric" Aβ species could provide the opportunity to produce some clinical benefits in AD subjects.

Accurate and timely flood forecasts are critical for making emergency-response decisions regarding public safety, infrastructure operations, and resource allocation. One of the main challenges for coastal flood forecasting systems is a lack of reliable forecast data of large-scale oceanic and watershed processes and the combined effects of multiple hazards, such as compound flooding at river mouths. Offshore water level anomalies, known as remote Non-Tidal Residuals (NTRs), are caused by processes such as downwelling, offshore wind setup, and also driven by ocean-basin salinity and temperature changes, common along the west coast during El Ni&ntilde, o events. Similarly, fluvial discharges can contribute to extreme water levels in the coastal area, while they are dominated by large-scale watershed hydraulics. However, with the recent emergence of reliable large-scale forecast systems, coastal models now import the essential input data to forecast extreme water levels in the nearshore. Accordingly, we have developed Hydro-CoSMoS, a new coastal forecast model based on the USGS Coastal Storm Modeling System (CoSMoS) powered by the Delft3D San Francisco Bay and Delta community model. In this work, we studied the role of fluvial discharges and remote NTRs on extreme water levels during a February 2019 storm by using Hydro-CoSMoS in hindcast mode. We simulated the storm with and without real-time fluvial discharge data to study their effect on coastal water levels and flooding extent, and highlight the importance of watershed forecast systems such as NOAA&rsquo, s National Water Model (NWM). We also studied the effect of remote NTRs on coastal water levels in San Francisco Bay during the 2019 February storm by utilizing the data from a global ocean model (HYCOM). Our results showed that accurate forecasts of remote NTRs and fluvial discharges can play a significant role in predicting extreme water levels in San Francisco Bay. This pilot application in San Francisco Bay can serve as a basis for integrated coastal flood modeling systems in complex coastal settings worldwide.

In the United States, Alzheimer's disease (AD) is the most common cause of dementia, accompanied by substantial economic and emotional costs. During 2015, more than 15 million family members who provided care to AD patients had an estimated total cost of 221 billion dollars. Recent studies have shown that elevated total plasma levels of homocysteine (tHcy), a condition known as hyperhomocysteinemia (HHcy), is a risk factor for AD. HHcy is associated with cognitive decline, brain atrophy, and dementia; enhances the vulnerability of neurons to oxidative injury; and damages the blood–brain barrier. Many therapeutic supplements containing vitamin B12 and folate have been studied to help decrease tHcy to a certain degree. However, a therapeutic cocktail approach with 5-methyltetrahydrofolate, methyl B12, betaine, and N-acetylcysteine (NAC) have not been studied. This novel approach may help target multiple pathways simultaneously to decrease tHcy and its toxicity substantially.

Skin cancer is by far the most common cancer, encompassing squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanoma. The diversity of cell types and tissue organisation in skin cancer remains poorly understood yet is required to improve diagnosis and treatment. In this work, we integrated six imaging and sequencing technologies to build the first spatial single cell reference for all three major skin cancer types and create a comprehensive skin cancer interactome. Using single-cell RNA-Seq (RNA) of >100,000 cells from 11 paired patient biopsies, we identified 28 SCC cell types, including 10 immune cell types, and found core suites of 39 cancer genes and 222 healthy genes shared across ≥80% patient samples. Using independent Nanostring Digital Spatial Profiling (RNA, protein), we validated most immune cell types and gene markers at protein and RNA levels. The enrichment of an immune signalling signature in SCC was further revealed by spatial Nanostring Single Molecular Imaging - SMI (RNA). Strikingly, we found the high consistency in mapping cell types in scRNAseq data and the independent SMI data, for example, the distribution of the three keratinocyte layers (basal, cycling and differentiated). This observation suggested the power of combining scRNAseq data with spatial SMI data. Furthermore, we implemented three approaches to validate the spatial distribution and cell type co-localisation by both Visium Spatial Transcriptomics (RNA), SMI (RNA) and Opal Multiplex Polaris (protein). Finally, cell-cell interactions were inferred at the global level using scRNAseq data (no spatial information) and Visium data (with spatial dimension), which were then validated at high throughput (517 ligands/receptors) and single-cell resolution using SMI. These in situ interaction maps were built across all three cancer types to create a comprehensive spatial interaction atlas of skin cancer. We also used targeted approaches with Polaris (protein) and RNAScope (RNA) to confirm and visualise clinically-important ligand-receptor pairs, including checkpoint inhibitor drug targets PD-1 and PD-L1. By integrating six distinct yet complementary spatial and single cell technologies, this study highlights the power of a spatial multi-omics approach for understanding cell types and their activities in cancer tissues. Citation Format: Laura Grice, Guiyan Ni, Xinnan Jin, Minh Tran, Emily Killingbeck, Mark Gregory, Onkar Mulay, Siok-Min Teoh, Arutha Kulasinghe, Michael Leon, Sarah Murphy, Sarah Warren, Youngmi Kim, Quan Nguyen. A single-cell, spatial multiomics atlas and cellular interactome of all major skin cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3817.

Current lithium-ion battery (LiB) recycling infrastructure is limited for strategic metals such as lithium and cobalt, despite projections that millions of electric vehicles (EVs) will hit the road in the next decade. Governments have labeled lithium and cobalt as “strategic” due to their importance in emerging green technologies as well as “critical” due to the increased risk of supply disruption resulting from geographic supply concentration, low substitutability, and low end-of-life recycling rates (EOL-RR’s). This paper aims to assist stakeholders conceptualize EV battery packs as a source of strategic metals and to help improve EOL-RR’s for lithium and cobalt. Specifically, this paper demonstrates the value of EV battery packs as a source of metals compared to natural resources and outlines advantages and disadvantages of thermal and mechanical recycling processes in terms of energy consumption, variable costs and maximum recoverable metal value. Findings suggest that EV battery packs contain favorable concentrations – often 1 magnitude higher – of lithium, cobalt, nickel and copper compared to respective economic ores. Disassembling the EV battery pack to the cell level increases cobalt, lithium and nickel concentrations since they are part of the cathode active material. Between thermal and mechanical recycling processes, the key tradeoff is between process time and ability to recover lithium. Thermal recycling requires less processing time than mechanical recycling, resulting in lower variable costs per battery pack, but mechanical recycling yields a higher maximum recoverable metal value since lithium can be recovered along with cobalt, copper and nickel.

We have suggested that reduced food intake increases the risk for anorexia nervosa by engaging mesolimbic dopamine neurons, thereby initially rewarding dieting. Recent fMRI studies have confirmed that dopamine neurons are activated in anorexia nervosa, but it is not clear whether this response is due to the disorder or to its resulting nutritional deficit. When the body senses the shortage of nutrients, it rapidly shifts behavior toward foraging for food as a normal physiological response and the mesolimbic dopamine neurons may be involved in that process. On the other hand, the altered dopamine status of anorexics has been suggested to result from a brain abnormality that underlies their complex emotional disorder. We suggest that the outcomes of the treatments that emerge from that perspective remain poor because they target the mental symptoms that are actually the consequences of the food deprivation that accompanies anorexia. On the other hand, a method that normalizes the disordered eating behavior of anorexics results in much better physiological, behavioral, and emotional outcomes.

We report the results of a study based on 1,428 patients with eating disorders treated at 6 clinics. These patients were consecutively referred over 18 years and used inpatient and outpatient treatment. The subjects were diagnosed with anorexia nervosa, bulimia nervosa, or an eating disorder not otherwise specified. Patients practiced a normal eating pattern with computerized feedback technology, they were supplied with external heat, their physical activity was reduced, and their social habits restored to allow them to return to their normal life. The estimated rate of remission for this therapy was 75% after a median of 12.5 months of treatment. A competing event such as the termination of insurance coverage, or failure of the treatment, interfered with outcomes in 16% of the patients, and the other patients remained in treatment. Of those who went in remission, the estimated rate of relapse was 10% over 5 years of follow-up and there was no mortality. These data replicate the outcomes reported in our previous studies and they compare favorably with the poor long-term remission rates, the high rate of relapse, and the high mortality rate reported with standard treatments for eating disorders.

Anorexia nervosa (AN) is classically defined as a condition in which an abnormally low body weight is associated with an intense fear of gaining weight and distorted cognitions regarding weight, shape, and drive for thinness. This article reviews recent evidences from physiology, genetics, epigenetics, and brain imaging which allow to consider AN as an abnormality of reward pathways or an attempt to preserve mental homeostasis. Special emphasis is put on ghrelino-resistance and the importance of orexigenic peptides of the lateral hypothalamus, the gut microbiota and a dysimmune disorder of neuropeptide signaling. Physiological processes, secondary to underlying, and premorbid vulnerability factors-the "pondero-nutritional-feeding basements"- are also discussed.

We examine the science and evidence supporting cognitive behavior therapy (CBT) for the treatment of bulimia nervosa and other eating disorders. Recent trials focusing on the abnormal cognitive and emotional aspects of bulimia have reported a remission rate of about 45%, and a relapse rate of about 30% within one year. However, an early CBT trial that emphasized the normalization of eating behavior had a better outcome than treatment that focused on cognitive intervention. In support of this finding, another treatment, that restores a normal eating behavior using mealtime feedback, has an estimated remission rate of about 75% and a relapse rate of about 10% over five years. Moreover, when eating behavior was normalized, cognitive and emotional abnormalities were resolved at remission without cognitive therapy. The critical aspect of the CBT treatment of bulimia nervosa therefore may actually have been the normalization of eating behavior.

In an effort to deepen our understanding of mammalian olfactory coding, we have used an objective method to analyze a large set of odorant-evoked activity maps collected systematically across the rat olfactory bulb to determine whether such an approach could identify specific glomerular regions that are activated by related odorants. To that end, we combined fuzzy c-means clustering methods with a novel validity approach based on cluster stability to evaluate the significance of the fuzzy partitions on a data set of glomerular layer responses to a large diverse group of odorants. Our results confirm the existence of glomerular response clusters to similar odorants. They further indicate a partial hierarchical chemotopic organization wherein larger glomerular regions can be subdivided into smaller areas that are rather specific in their responses to particular functional groups of odorants. These clusters bear many similarities to, as well as some differences from, response domains previously proposed for the glomerular layer of the bulb. These data also provide additional support for the concept of an identity code in the mammalian olfactory system.

IOAKIMIDIS I, M. ZANDIAN, F. ULBL, C. BERGH, M LEON, AND P. SÖDERSTEN. How eating affects mood. PHYSIOL BEHAV 2011 (000) 000-000. We hypothesize that the changes in mood that are associated with eating disorders are caused by a change in eating behavior. When food is in short supply, the rhythm of the neural network for eating, including orbitofrontal cortex and brainstem, slows down and we suggest that this type of neural activity activates a partially overlapping neural network for mood, including dorsal raphe serotonin projections to the orbitofrontal and prefrontal cortex. As a consequence, people who restrict the amount of food that they consume, either by choice or by their limited access to food, become preoccupied with food and food-related behavior. Most eating disorders emerge from a history of dietary restriction and we suggest that disordered eating consequent upon food restriction produces the altered mental state of patients with eating disorders. Based on the present hypothesis, eating disorders are not the result of a primary mental disorder. Rather, this notion suggests that the patients should be treated by learning to eat an appropriate amount of food at an appropriate rate.

Although it has been shown repeatedly that minimum response times in sensory systems can be quite short, organisms more often continue to respond to sensory stimuli over considerably longer periods of time. The continuing response to sensory stimulation may be a more realistic assessment of natural sensory responses, so we determined for how long a stimulus would evoke a response in naïve, freely moving animals. Specifically, we determined for how long such rats responded to odorants during continuous passive exposures by monitoring their sniffing with whole-body plethysmography. We found that naïve rats continue to sniff odorants vigorously for up to 3 minutes, much longer than what has been reported for highly trained, highly motivated rats. Patterns of 2-deoxyglucose (2-DG) uptake in the glomerular layer of the rat olfactory bulb also were seen after only 1-5 minutes of odorant exposure, overlapping with the period of increased respiration to odorants. Moreover, these 2-DG uptake patterns closely resembled the patterns that emerge from prolonged odorant exposures, suggesting that activity mapping over prolonged periods can identify areas of activity that are present when rats are still attending and responding to odorant stimuli. Given these findings, it seems important to consider the possibility that prolonged exposure to other sensory stimuli will reveal more realistic neural response patterns.

The coding of olfactory stimuli across a wide range of organisms may rely on fundamentally similar mechanisms in which a complement of specific odorant receptors on olfactory sensory neurons respond differentially to airborne chemicals to initiate the process by which specific odors are perceived. The question that we address in this review is the role of specific neurons in mediating this sensory system--an identity code--relative to the role that temporally specific responses across many neurons play in producing an olfactory perception--a temporal code. While information coded in specific neurons may be converted into a temporal code, it is also possible that temporal codes exist in the absence of response specificity for any particular neuron or subset of neurons. We review the data supporting these ideas, and we discuss the research perspectives that could help to reveal the mechanisms by which odorants become perceptions.

One of the salient characteristics of the United States Social Forum (USSF) was its diversity. The driving force behind the organizing of the USSF was a sector of grassroots organizations largely overlooked in national politics. This grassroots movement represents the potential for new political direction and hope for fundamental change in the US. The World Social Forum is a global phenomenon that was conceived through important historical precedents. Global Justice Alliance (GGJ) was created to provide capacity for the grassroots sector to be proactive in its participation internationally and to strengthen national grassroots movement building in the United States. The USSF was testimony to the resiliency of the people who make up the grassroots Global Justice Movement (GJM) in the United States - the poor, the marginalized, the unrepresented. Keywords: Global Justice Alliance (GGJ); Global Justice Movement (GJM); grassroots movement; United States Social Forum (USSF)

We have previously shown in two randomized clinical trials that environmental enrichment is capable of ameliorating symptoms of autism spectrum disorder (ASD), and in the present study, we determined whether this therapy could be effective under real-world circumstances. 1,002 children were given daily Sensory Enrichment Therapy, by their parents, using personalized therapy instructions given over the Internet. Parents were asked to assess the symptoms of their child every 2 weeks for up to 7 months. An intention-to-treat analysis showed significant overall gains for a wide range of symptoms in these children, including learning, memory, anxiety, attention span, motor skills, eating, sleeping, sensory processing, self-awareness, communication, social skills, and mood/autism behaviors. The children of compliant caregivers were more likely to experience a significant improvement in their symptoms. The treatment was effective across a wide age range and there was equal progress reported for males and females, for USA and international subjects, for those who paid and those who did not pay for the therapy, and for individuals at all levels of initial symptom severity. Environmental enrichment, delivered via an online system, therefore appears to be an effective, low-cost means of treating the symptoms of ASD.