The Role of Hypothalamic Phospholipid Liposomes in the Supportive Therapy of Some Manifestations of Long Covid: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Brain Fog (preprint)

Long Covid is a heterogeneous clinical condition in which Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and brain fog stand out among the different clinical symptoms and syndromes. The cerebral metabolic alterations and neuroendocrine disorders seem to constitute an important part of Long Covid. Given the substantial lack of drugs and effective therapeutic strategies, hypothalamic phospholipid liposomes which have been on the market for several years as adjuvant therapy of cerebral metabolic alterations resulting from neuroendocrine disorders, can be taken into consideration in an overall therapeutic strategy that aims to control the Long Covid associated symptoms and syndromes. Their pharmacological mechanisms and clinical effects strongly support their usefulness in Long Covid. Our initial clinical experience corroborates this rationale. Further research is imperative in order to obtain robust clinical evidence.

Pituitary Diseases and COVID-19 Outcomes in South Korea: A Nationwide Cohort Study (preprint)

Background The pituitary gland is either directly or indirectly impacted by SARS-CoV-2 infection. As a consequence of SARS-CoV-2 infection, hypothalamic-pituitary dysfunction or pituitary apoplexy can occur. This study aimed to investigate severe COVID-19 outcomes and COVID-19-related mortality in patients with underlying pituitary disease in Korea using a nationwide cohort database.Methods Data required for this study were obtained from the Health Insurance Review and Assessment Service of Korea. Patients with SARS-CoV-2 infection between January 2020 and December 2021 were divided into the following three groups and analyzed: Group A, those who were hospitalized for SARS-CoV-2 infection without underlying pituitary disease (n = 725,170); Group B, those who were hospitalized for SARS-CoV-2 infection with underlying pituitary disease without exposure to systemic steroids (n = 1,509); Group C, patients with underlying pituitary disease and exposure to systemic steroids (n = 365). Differences in severe COVID-19, requirement for oxygen therapy, intensive care unit admission, application of invasive ventilation or use of extracorporeal membrane oxygenation, and COVID-19-related death between groups were then analyzed.Results Group C had the highest rates of hospitalization after COVID-19 infection (82.2%) and mortality within 30 days of infection (6.8%). Group B had 1.3-fold increase in severe COVID-19 outcome compared to Group A. Group C had 1.8-fold and 1.3-fold increase in severe COVID-19 outcome compared to Group A and Group B, respectively. Group C also had 2.34 and 3.24 times higher mortality rate within 30 days of COVID-19 infection than in Group A and Group B, respectively.Conclusion In conclusion, patients with pituitary disease who are receiving systemic steroids have poorer outcomes and higher mortality associated with COVID-19. Therefore, thorough COVID-19 infection control is required in these patients.

Modification of Sympathetic and Hypothalamic Responses to Prevent Complications of COVID-19 “Dam and Wall Concept” (preprint)

We are in amidst of COVID-19 pandemic. Since Dec 2019, severe acute respiratory corona virus (SAR-CoV-2) has infected more than half a billion people killing nearly 7 million people worldwide. Now the BA.5 variant of SARS-CoV-2 is causing mayhem and driving the global surge. Epidemiologist are aware of the fact that this virus is capable of escaping immunity and likely to infect the same person multiple times despite adequate vaccination status. Elderly people of age more than 60 years and those with underlying health conditions are considered as high-risk who are likely to suffer complications and death. While it is tempting to frame complications and mortality from COVID-19 as a simple matter of too much of a virulent virus in too weak of a host, much more is at play here. Framing the pathophysiology of COVID-19 in the context of the Chrousos and Gold model of the central stress response system can shed insight into its complex pathogenesis. Understanding the mechanisms by which pharmacologic modulation of the central stress response system via administration of clonidine and/or dexamethasone may offer an explanation as to why a viral pathogen can be well tolerated and cleared by one host while inflaming and killing another.

A missense mutation in zinc finger homeobox-3 (ZFHX3) impedes growth and alters metabolism and hypothalamic gene expression in mice (preprint)

Objective A protein altering variant in the gene encoding zinc finger homeobox-3 ( ZFHX3 ) has recently been associated with lower BMI in a human genome-wide association study. We investigated metabolic parameters in mice harbouring a missense mutation in Zfhx3 ( Zfhx3 Sci/+ ) and looked for altered expression of transcripts that are associated with energy balance in the hypothalamus to understand how ZFHX3 may influence metabolic effects. Methods Body weight, length and composition were measured in 1 year old male and female Zfhx3 Sci/+ and Zfhx3 +/+ mice, and fat depot weights were measured along with fasted anabolic hormone concentrations. In a second cohort of male and female Zfhx3 Sci/+ and Zfhx3 +/+ mice weekly food intake was measured from the age of 9 – 20 weeks. A third cohort of female-only Zfhx3 Sci/+ and Zfhx3 +/+ mice underwent body composition analysis every two weeks from 6 - 14 weeks of age, and 24 h energy expenditure was assessed. At 16 weeks old, brains were collected in the light and dark phases for mRNA in situ hybridisation analysis of candidate genes identified in previous RNA-seq analysis of hypothalamic tissue of Zfhx3 Sci/+ mice. Results We found that 1 year old Zfhx3 Sci/+ mice weighed less and had a shorter body length than wildtype littermates, and had reduced fat mass, smaller mesenteric fat depots, and lower circulating insulin, leptin and insulin-like growth factor-1 (IGF1) concentrations. Male and female Zfhx3 Sci/+ mice ate less than wildtype controls from 10 weeks of age. In the final experiment, female Zfhx3 Sci/+ mice weighed less and had lower lean mass, but fat mass didn’t differ. Energy expenditure was lower in Zfhx3 Sci/+ mice. We detected increased expression of somatostatin, and decreased expression of growth hormone-releasing hormone and growth hormone-receptor mRNAs in the arcuate nucleus (ARC), but these transcripts were unaltered in other hypothalamic nuclei. Similarly, ARC expression of orexigenic neuropeptide Y was decreased and ventricular ependymal layer expression of orphan G protein-coupled receptor Gpr50 was decreased. Conclusions The Sci missense mutation conferred a protective metabolic phenotype in mice and implied a somatic growth effect. The promoters of mouse Sst and Gpr50 genes contain ZFHX3 binding AT-motifs, suggesting growth axis effects are driven by transcriptional activation of Sst in the ARC. It is likely that altered Gpr50 gene expression led to decreased energy expenditure and decreased Npy expression in the ARC decreased appetite. Together these results point to a pleiotropic effect of ZFHX3 in regulating energy balance, independent of its circadian effects. Highlights - Male and female Zfhx3 Sci/+ mice have a shorter body length, eat less and weigh less - Zfhx3 Sci/+ mice have lower insulin, leptin and IGF1 in circulation - Zfhx3 Sci/+ mice have decreased Ghr, Ghrh and Npy expression in the arcuate nucleus - Arcuate nucleus expression of AT-motif containing Sst is increased in Zfhx3 Sci/+ mice - This model may explain human protein altering ZFHX3 variant associated with low BMI

Assessment of Neuroendocrine Changes and the Hypothalamo-Pituitary Autoimmunity in Patients With COVID-19 (preprint)

Purpose: The SARS-CoV-2 may affect the hypothalamic-pituitary axis and pituitary dysfunction may occur. Therefore, we investigated neuroendocrine changes, particularly, secondary adrenal insufficiency using a dynamic test and the role of autoimmunity in pituitary dysfunction in the patients with COVID-19. Methods: : The single-center, prospective, case-control study included PCR-confirmed COVID-19 patients and healthy controls. Basal hormone levels were measured and ACTH stimulation test was performed. Anti-pituitary (APA) and anti-hypothalamic antibodies (AHA) were also determined. Results: : We examined a total of 49 patients with COVID-19 and 28 healthy controls. The frequency of adrenal insufficiency in patients with COVID-19 was found as 8.2%. The patients with COVID-19 had lower free T 3 , IGF-1, total testosterone levels, and higher cortisol and prolactin levels when compared with controls. We also, demonstrated the presence of APA in three and AHA in one of four patients with adrenal insufficiency. Conclusions: : The COVID-19 may result in adrenal insufficiency, so the routine screening of adrenal functions is these patients is needed. Endocrine disturbances in COVID-19 are similar to those seen in acute stressful conditions or infections. Also, pituitary or hypothalamic autoimmunity may play a role in neuroendocrine abnormality in COVID-19.

Brain thermal kinetics at brain-eyelid thermal tunnels overcoming COVID-19 thermometry limitations for automated asymptomatic infection detection in concert with physical and biological principles (preprint)

For centuries, temperature measurement deficiencies attributable to biological barriers and low thermo-conductivity ( k ) have precluded accurate surface-based fever assessment. At this stage of the pandemic, infection detection in children (who due to immature immune system may not effectively respond to vaccines) is critical because children can be readily infected and also become a large mutation reservoir. We reveal hitherto-unrecognized worldwide body temperature measurements (T°), in children and adults, over tissue typified by low- k  similar to wood that may reach 6.8°C in thermal variability, hampering thereby COVID-19 control. Brain-eyelid thermal tunnels’ (BTT) integration of low- k  and high- k  regions creating a thermal pathway for undisturbed heat transmission from hypothalamus to high- k  skin eliminates current shortcomings and makes the brain indispensable for defeating COVID-19 given that brain thermoregulatory signals are not limited by mutations. Anatomo-histologic, emissive, physiologic, and thermometric bench-to-bedside studies characterized and overcome biophysical limitations of thermometry through high- k  eyelid-enabled brain temperature measurements in children and adults. BTT eyelid features fat-free skin (~900 µm) and unique light emission through a blood/fat configuration in the underlying tunnel. Contrarily, forehead features variable and thick dermis (2000–2500 µm) and variable fat layers (1100–2800 µm) resulting in variable low- k  as well as temperatures 1.97 °C lower than BTT temperature (BTT°). Highest emission present in only ~3.1% of forehead averaged 1.08±0.49 °C (mean±SD) less than BTT° ( p =0.008). Environmental and biological impacts during fanning revealed thermal imaging limitations for COVID-19 screening. Comparison of paired measurements for 100 pediatric patients showed that in the children subgroup above 37°C, BTT° exceeded body core temperature (Core°) in 60/72 patients; the average difference in the 72 patients was 0.62±0.7°C  (p<0.001 by unpaired t-test); and in the subgroup beyond 37.5°C, BTT° exceeded Core° in 30/32 patients. Delineating hypothalamic activity in children facilitates early infection detection, which is essential because children’s immunogenicity prevents effective vaccination and causes accelerated viral evolution. Capturing hypothalamic thermal signals from BTT was further supported by brain thermal kinetics via BTT using wearables during anesthesia, sedation, sleep, brain injury, exercise, and asymptomatic infection, which revealed brain/core discordance and enabled automated noninvasive afebrile infection detection for interrupting asymptomatic human-to-human transmission. BTT-based spot-check thermometry can be harmlessly implemented for children worldwide without undue burden and costs; meanwhile, continuous brain-eyelid T° in concert with biological and physical principles affords a new dimension for combating pandemics. The “detection–vaccination” pair solution presented is required to mitigate COVID-19 from spreading indefinitely through mutations and vaccine evasion while opening a viable path for eradicating COVID-19.

A Case Report on Steroid-Induced Avascular Necrosis Treated with Autologous Adult Live-Cultured Osteoblasts (AALCO) in COVID-19 Patients (preprint)

In the absence of a targeted antiviral agent effective against COVID-19, corticosteroids show improvements in hyperinflammation and acute respiratory distress syndrome (ARDS) associated with COVID-19. However, various adverse effects of corticosteroids have been reported, like delayed viral clearance, secondary infections, avascular necrosis and suppression of the hypothalamic-pituitary-adrenal axis. In this case report, we report clinical outcomes in two patients with symptomatic avascular necrosis (AVN) of the femoral head after being treated with corticosteroids for COVID-19. We implanted autologous adult live-cultured osteoblasts (AALCO), sourced from mesenchymal stem cells via bone marrow aspiration, for treating AVN in both patients. The MRI examination suggested steroid-induced AVN of the hip with Ficat-Arlet stage IIB in these patients. A prednisone (corticosteroid) was used during COVID-19 treatment for a mean treatment duration of 14.5 days. The AALCO implantation showed good clinical outcomes in treating steroid-induced AVN of the hip with Ficat-Arlet stage IIB in both patients with COVID-19.

SC-MEB: spatial clustering with hidden Markov random field using empirical Bayes (preprint)

Spatial transcriptomics has been emerging as a powerful technique for resolving gene expression profiles while retaining tissue spatial information. These spatially resolved transcriptomics make it feasible to examine the complex multicellular systems of different microenvironments. To answer scientific questions with spatial transcriptomics and expand our understanding of how cell types and states are regulated by microenvironment, the first step is to identify cell clusters by integrating the available spatial information. Here, we introduce SC-MEB, an empirical Bayes approach for spatial clustering analysis using a hidden Markov random field. We have also derived an efficient expectation-maximization algorithm based on an iterative conditional mode for SC-MEB. In contrast to BayesSpace, a recently developed method, SC-MEB is not only computationally efficient and scalable to large sample sizes but is also capable of choosing the smoothness parameter and the number of clusters. We performed comprehensive simulation studies to demonstrate the superiority of SC-MEB over some existing methods. We applied SC-MEB to analyze the spatial transcriptome of human dorsolateral prefrontal cortex tissues and mouse hypothalamic preoptic region. Our analysis results showed that SC-MEB can achieve a similar or better clustering performance to BayesSpace, which uses the true number of clusters and a fixed smoothness parameter. Moreover, SC-MEB is scalable to large ‘sample sizes’. We then employed SC-MEB to analyze a colon dataset from a patient with colorectal cancer (CRC) and COVID-19, and further performed differential expression analysis to identify signature genes related to the clustering results. The heatmap of identified signature genes showed that the clusters identified using SC-MEB were more separable than those obtained with BayesSpace. Using pathway analysis, we identified three immune-related clusters, and in a further comparison, found the mean expression of COVID-19 signature genes was greater in immune than non-immune regions of colon tissue. SC-MEB provides a valuable computational tool for investigating the structural organizations of tissues from spatial transcriptomic data.

ACE2 expression in rat brain: implications for COVID-19 associated neurological manifestations (preprint)

We examined cell type-specific expression and distribution of rat brain angiotensin converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, in rodent brain. ACE2 is ubiquitously present in brain vasculature, with the highest density of ACE2 expressing capillaries found in the olfactory bulb, the hypothalamic paraventricular, supraoptic and mammillary nuclei, the midbrain substantia nigra and ventral tegmental area, and the hindbrain pontine nucleus, pre-Botzinger complex, and nucleus of tractus solitarius. ACE2 was expressed in astrocytes and astrocytic foot processes, pericytes and endothelial cells, key components of the blood-brain-barrier. We found discrete neuronal groups immunopositive for ACE2 in brainstem respiratory rhythm generating centers including the pontine nucleus, the parafascicular/retrotrapezoid nucleus, the parabrachial nucleus, the Botzinger and pre-Botzinger complex and the nucleus of tractus solitarius; in arousal-related pontine reticular nucleus and in gigantocellular reticular nuclei; in brainstem aminergic nuclei, including substantia nigra, ventral tegmental area, dorsal raphe, and locus coeruleus; in the epithalamic habenula, hypothalamic paraventricular and suprammamillary nuclei; and in the hippocampus. Identification of ACE2-expressing neurons in rat brain within well-established functional circuits facilitates prediction of possible neurological manifestations of brain ACE2 dysregulation during and after COVID-19 infection.

Effects of the COVID-19 pandemic and lockdown on the mental and physical health of adults with Prader-Willi Syndrome (preprint)

Background Prader-Willi syndrome (PWS) is a neurodevelopmental disorder with hypothalamic dysfunction leading to obesity and behavioral disabilities, including eating disorders (EDs). We evaluated the effects of the COVID-19 infection and lockdown on mental and physical health in PWS. At the end of April, 85 adults with PWS completed a self-administered questionnaire, including lockdown conditions, physical activity (PA), ED, and medical and behavioral outcomes. Body weight was measured at home and self-reported. Results Patients (53% women, 45% disomic) were assessed, with a mean age of 28.1±8.7 years and body mass index (BMI) of 37±11 kg/m². Seventy percent lived in the Paris region (France) and were confined with their parents. The mean weight change was 1.0±3.3 kg. We compared patients showing weight loss (n=39, -3.3±2.9 kg) to patients showing weight gain (n=22, +2.3±1.5 kg): the BMI was lower (34.6±9.2 versus 40.4±9.5 kg/m², p=0.02), PA increased (26% versus 5%, p=0.04), and EDs improved (51% versus 14%, p=0.005). Behavioral disorders increased for 13% of the cohort. Three individuals (3.5%) were diagnosed with non-severe COVID-19. Conclusion Lockdown during the COVID-19 pandemic was associated with positive effects for most French adults with PWS, with weight loss probably associated with a more favourable environment during this period. We observed no severe forms of COVID-19.

The hypothalamus as a hub for putative SARS-CoV-2 brain infection (preprint)

Most patients with COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), display neurological symptoms, and respiratory failure in certain cases could be of extra-pulmonary origin. Hypothalamic neural circuits play key roles in sex differences, diabetes, hypertension, obesity and aging, all risk factors for severe COVID-19, besides being connected to olfactory/gustative and brainstem cardiorespiratory centers. Here, human brain gene-expression analyses and immunohistochemistry reveal that the hypothalamus and associated regions express angiotensin-converting enzyme 2 and transmembrane proteinase, serine 2, which mediate SARS-CoV-2 cellular entry, in correlation with genes or pathways involved in physiological functions or viral pathogenesis. A post-mortem patient brain shows viral invasion and replication in both the olfactory bulb and the hypothalamus, while animal studies indicate that sex hormones and metabolic diseases influence this susceptibility.