Autopsy Tissue Research Articles

Ultrastructural evidence for vertical transmission of SARS-CoV-2

Since the initial correspondence by Goldsmith et al. (2020) in The Lancet, numerous electron micrographs of putative severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virions in biopsy and autopsy tissues have been published. A recent review of these images (Bullock et al., 2021) indicated that previous ultrastructural reports of virions in placental tissue were misidentifications. Placental histology of mothers and neonates, both testing positive for SARS-CoV-2, is typified by chronic histiocytic intervillositis and trophoblast necrosis, with RNA in-situ hybridization/immunohistochemical findings localizing viral RNA/viral antigens to the syncytiotrophoblast (Schwartz and Morotti, 2020).

Elevated Oxidative Stress and DNA Damage in Cortical Neurons of Chemotherapy Patients.

The unintended neurologic sequelae of chemotherapy contribute to significant patient morbidity. Chemotherapy-related cognitive impairment (CRCI) is observed in up to 80% of cancer patients treated with chemotherapy and involves multiple cognitive domains including executive functioning. The pathophysiology underlying CRCI and the neurotoxicity of chemotherapy is incompletely understood, but oxidative stress and DNA damage are highly plausible mechanisms based on preclinical data. Unfortunately, validating pathways relevant to CRCI in humans is limited by an absence of relevant neuropathologic studies of patient brain tissue. In the present study, we stained sections of frontal lobe autopsy tissue from cancer patients treated with chemotherapy (n = 15), cancer patients not treated with chemotherapy (n = 10), and patients without history of cancer (n = 10) for markers of oxidative stress (nitrotyrosine, 4-hydroxynonenal) and DNA damage (pH2AX, pATM). Cancer patients treated with chemotherapy had increased staining for markers of oxidative stress and DNA damage in frontal lobe cortical neurons compared to controls. We detected no statistically significant difference in oxidative stress and DNA damage by the duration between last administration of chemotherapy and death. The study highlights the potential relevance of oxidative stress and DNA damage in the pathophysiology of CRCI and the neurotoxicity of chemotherapy.

Propagation of Activated B Cells by In Vitro Severe Fever With Thrombocytopenia Syndrome Virus Infection of Human Peripheral Blood Mononuclear Cells.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging, life-threatening tick-borne viral hemorrhagic fever caused by SFTS virus (SFTSV). Transient appearance of plasmablastic lymphocytes in the peripheral blood of SFTS cases has been reported; however, the pathological significance of this transient burst in peripheral blood plasmablastic lymphocytes is unclear. Here, we show that SFTSV infection of human peripheral blood mononuclear cells in vitro induced propagation of atypical lymphocytes. These atypical lymphocytes were activated B cells, which were induced by secretory factors other than viral particles; these factors were secreted by SFTSV-infected B cells. Activated B cells shared morphological and immunophenotypic characteristics with B cells of plasmablast lineage observed in peripheral blood and autopsy tissues of SFTS cases. This suggests that SFTSV-infected B cells secrete factors that induce B-cell differentiation to plasmablasts, which may play an important role in pathogenesis of SFTS through the SFTSV-B cell axis.

Semi-Automated Determination of Heavy Metals in Autopsy Tissue Using Robot-Assisted Sample Preparation and ICP-MS

The endoprosthetic care of hip and knee joints introduces multiple materials into the human body. Metal containing implant surfaces release degradation products such as particulate wear and corrosion debris, metal-protein complexes, free metallic ions, inorganic metal salts or oxides. Depending on the material composition of the prostheses, a systemic exposure occurs and may result in increasing metal concentrations in body fluids and tissues especially in the case of malfunctions of the arthroplasty components. High concentrations of Cr, Co, Ni, Ti and Al affect multiple organs such as thyroid, heart, lung and cranial nerves and may lead to metallosis, intoxications, poly-neuropathy, retinopathy, cardiomyopathy and the formation of localized pseudo tumors. The determination of the concentration of metals in body fluids and tissues can be used for predicting failure of hip or knee replacements to prevent subsequent severe intoxications. A semi-automated robot-assisted measurement system is presented for the determination of heavy metals in human tissue samples using inductively coupled plasma mass spectrometry (ICP-MS). The manual and automated measurement processes were similarly validated using certified reference material and the results are compared and discussed. The automation system was successfully applied in the determination of heavy metals in human tissue; the first results are presented.

Viral infiltration of pancreatic islets in patients with COVID-19

Metabolic diseases are associated with an increased risk of severe COVID-19 and conversely, new-onset hyperglycemia and complications of preexisting diabetes have been observed in COVID-19 patients. Here, we performed a comprehensive analysis of pancreatic autopsy tissue from COVID-19 patients using immunofluorescence, immunohistochemistry, RNA scope and electron microscopy and detected SARS-CoV-2 viral infiltration of beta-cells in all patients. Using SARS-CoV-2 pseudoviruses, we confirmed that isolated human islet cells are permissive to infection. In eleven COVID-19 patients, we examined the expression of ACE2, TMPRSS and other receptors and factors, such as DPP4, HMBG1 and NRP1, that might facilitate virus entry. Whereas 70% of the COVID-19 patients expressed ACE2 in the vasculature, only 30% displayed ACE2-expression in beta-cells. Even in the absence of manifest new-onset diabetes, necroptotic cell death, immune cell infiltration and SARS-CoV-2 viral infection of pancreatic beta-cells may contribute to varying degrees of metabolic dysregulation in patients with COVID-19.

Measurement of treatment dependent glioblastoma cell density in T1- weighted contrast enhancement at autopsy.

e14035 Background: With an average overall survival of 12-18 months, glioblastoma has a particularly grim diagnosis. Standard treatment of glioblastoma, following detection on MRI, is surgical resection followed by radiation therapy and chemotherapy and is monitored through MR imaging. Glioblastoma has a unique heterogenous nature that complicates visualization of subtly enhancing tumor. This study used autopsy tissue samples taken from glioblastoma patients with varying treatment, to examine the effects of treatment on cell density within regions of contrast enhancement, using T1-weighted subtraction maps (T1S) from the last MR images to death. Methods: Eight patients diagnosed with glioblastoma at autopsy were recruited for this study. Two patients had no treatment and six received a combination of chemo-radiation and other treatments, including but not limited to bevacizumab (Bev) and TTFields therapy. At autopsy, whole brain samples were sliced axially aligned to the patient’s final MRI to death. Time between last MRI and death ranged from 4-27 days (mean 16 days). Overall survival (OS) ranged from 4-538 days (mean 307 days). Large tissue samples were taken from regions of suspected tumor or treatment effect, for a total of 18 tissue samples. Tissue samples were processed, H&E stained, and digitized at 40X resolution (Huron Slide Scanner). Cell density (cells/mm2) was calculated using digital histology. T1S were created for each patient by subtracting intensity normalized T1 weighted images from T1 post contrast images (T1C). Digital histology was aligned and resampled into MRI space using manual control point registration. Mixed effect models were used to compare differences in cell density across contrast enhancement (T1SE vs. NE) as well as across treatment groups (treatment vs. no treatment). Results: Cellularity was compared across regions of T1S enhancement (T1SE) and non-enhancement (NE) within manually selected regions of interest. Cell density compared between regions of T1SE and NE was not different (p=0.219). Total cell density was increased in patients who had received treatment compared to no treatment in both regions of T1SE and NE (p=0.014). Conclusions: Overall, cell density was increased in patients who had received treatment after diagnosis of glioblastoma. Additional research is needed to examine the extent of treatment’s effect on cellularity of glioblastoma. This work begins to characterize the use of T1S in evaluating glioblastoma tumor burden in patients with varying treatment histories.[Table: see text]

Autoimmune Global Amnesia as Manifestation of AMPAR Encephalitis and Neuropathologic Findings.

ObjectiveTo report an unusual clinical phenotype of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) encephalitis and describe associated neuropathologic findings.MethodsWe retrospectively investigated 3 AMPAR encephalitis patients with autoimmune global hippocampal amnesia using comprehensive cognitive and neuropsychologic assessment, antibody testing by in-house tissue-based and cell-based assays, and neuropathologic analysis of brain autopsy tissue including histology and immunohistochemistry.ResultsThree patients presented with acute-to-subacute global amnesia without affection of cognitive performance, attention, concentration, or verbal function. None of the patients had epileptic seizures, change of behavior, personality changes, or psychiatric symptoms. The MRI was normal in 1 patient and showed increased fluid-attenuated inversion recovery/T2 signal in the hippocampus in the other 2 patients. Two patients showed complete remission after immunotherapy. The one patient who did not improve had an underlying adenocarcinoma of the lung and died 3.5 months after disease onset because of tumor progression. Neuropathologic analysis of the brain autopsy revealed unilateral hippocampal sclerosis accompanied by mild inflammatory infiltrates, predominantly composed of T lymphocytes, and decrease of AMPAR immunoreactivity.ConclusionAMPAR antibodies usually associate with limbic encephalitis but may also present with immune responsive, acute-to-subacute, isolated hippocampal dysfunction without overt inflammatory CSF or MRI changes.

Point of care real-time polymerase chain reaction-based diagnostic for Kyasanur forest disease

ObjectivesDue to the remote forest area locations of sporadic cases and outbreaks of Kyasanur forest disease (KFD), rapid diagnosis poses a significant challenge. This study aimed to evaluate the diagnostic performance of Truenat KFD, a simple, rapid and user-friendly point-of-care test for detection of KFD and compare diagnostic accuracy with conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) testing. Truenat KFD can be deployed in a field laboratory setting. MethodsThe study involved 145 clinical specimens, including human serum, monkey necropsy tissues and tick pool, to validate Truenat KFD (Molbio Diagnostics Pvt.Ltd.) for KFD diagnosis. ResultsWe have optimized and validated the microchip-based Truenat KFD (Molbio Diagnostics Pvt.Ltd.) for KFD diagnosis. Point-of-care testing was highly sensitive and specific, with a detection limit of up to 10 copies of KFD viral RNA. Results were comparable with the gold-standard TaqMan and commercially available Altona RealStar AHFV / KFDV real-time RT-PCR assays. Screening results for human, monkey and tick specimens were 100% concordant across the assays. ConclusionTruenat KFD(Molbio Diagnostics Pvt.Ltd.) was found to be highly sensitive and specific with a significant limit of detection. This point-of-care test would be useful in rapid diagnosis of KFD in remote and/or field settings, quick patient management and control of virus spread.

Proteome analysis demonstrates involvement of endoplasmic reticulum stress response in human myocardium with subclinical left ventricular diastolic dysfunction.

Heart failure is increasing in Japan, in particular that with preserved ejection fraction (HFpEF) prevalent in older-aged patients. The purpose of this study was to investigate the pathophysiology during the early stage of left ventricular (LV) diastolic dysfunction by the quantitative proteome analysis of human myocardium. Among 331 post-mortem autopsy patients, we selected 23 patients (aged 79 ± 9.6 years) with echocardiographic data and without major comorbidities, except hypertension. Cryopreserved autopsy tissue of the LV myocardium was subjected to proteome analysis. LV diastolic function was evaluated by echocardiographic data. Thirteen patients were classified into the impaired diastolic function (IDF) group, and 10 the normal cardiac function group. We performed comparative proteome analysis between the IDF and normal groups by isobaric tags for relative and absolute quantitation (iTRAQ) using nano-liquid chromatography-tandem mass spectrometry. The iTRAQ-based proteome analysis revealed 57 differentially expressed proteins in the IDF group. Molecular network analysis of differentially expressed proteins indicated that endoplasmic reticulum (ER) stress was a potentially important event. Furthermore, the expressions of proteins associated with the ER stress response, such as glucose-regulated protein 78 kDa, inositol-requiring kinase 1α and spliced X-box binding protein 1, were significantly decreased in the IDF group. This study suggested that reduced ER stress responses were involved during the early stage of LV diastolic dysfunction. Geriatr Gerontol Int ••; ••: ••-•• Geriatr Gerontol Int 2021; 21: 577-583.

Native Endogenous Fluorescence Imaging Detects Vascular Occlusions in Patients with COVID-19

Background: A number of histopathological reports showed the presence of widespread thrombosis and associated morphology in pulmonary vessels of patients with COVID-19. Later, we identified vascular occlusions with neutrophils and neutrophil extracellular traps (NETs), as major components, in autopsy tissue from patients with COVID-19.Methods: We, here investigated 109 lung specimens from 17 patients with COVID-19 and compared them with 11 lung specimens from two patients who succumbed to pulmonary embolism and acute cardiac. Healthy lung specimens from four patients served as controls. We studied these autopsy lung specimens using immunohistochemistry and native endogenous fluorescence.Findings: We present a label-free imaging technique using native endogenous fluorescence that enables the visualization of occluded vessels. We demonstrate that native endogenous fluorescence identified occluded vessels in tissue specimens from patients with COVID-19.Interpretation: Label-free detection of occluded vessels enabled the detection of affected occluded vessels in lung specimens of patients with COVID-19 where the occluding components showed varying contents of neutrophil-derived materials.Funding Statement: Deutsche Forschungsgemeinschaft (DFG), EU, Volkswagen-StiftungDeclaration of Interests: All authors have no conflicts of interests to declare.Ethics Approval Statement: An institutional approval from each local Ethical Committee was obtained (permit #193_13B; permit # 174_20B; EK 092/20; EK 119/20; EK 460/20).

Acute and Sub-chronic Toxicity Study of Recombinant Bovine Interferon Alpha in Rodents.

IntroductionRecombinant bovine interferon alpha (rBoIFN-α) has been demonstrated to have antiviral activity. However, no conduct of acute or chronic toxicity tests has been reported.Material and MethodsSpecific pathogen-free Sprague Dawley rats were administered doses at different concentrations through intraperitoneal or intravenous injection. After the administration (single for an acute toxicity test over 14 days or daily for a sub-chronic toxicity test over 30 days), the rats’ behaviour and other indicators and the degree of toxic reaction were continuously monitored. Blood was collected for haematological and serum biochemical examinations. At the end of the experiments, the rats were sacrificed for necropsy and histopathological tissue analysis.ResultsThe external performance, behaviour characteristics, and changes in body temperature and body weight of the rats in each subgroup were comparable to the normal control subgroup. Except for a few cases, there were no lesions in the viscera’s pathological structures, and the blood parameters and biochemical indicators were not noticeably different from those of the control subgroup.ConclusionThis study suggests that rBoIFN-α seems to be safe for rats, and its use may foster the development of the cattle industry in China by protecting livestock health.

Risk Assessment of Perfluorooctane Sulfonate (PFOS) using Dynamic Age Dependent Physiologically based Pharmacokinetic Model (PBPK) across Human Lifetime

The widespread use of Perfluorooctane sulfonate (PFOS) in everyday life, its long half-life, and the lipophilicity that makes it easily accumulate in the body, raises the question of its safe exposure among different population groups. There are currently enough epidemiological studies showing evidence of PFOS exposure and its associated adverse effects on humans. Moreover, it is already known that physiological changes along with age e.g. organ volume, renal blood flow, cardiac output and albumin concentrations affect chemicals body burden. Human biomonitoring cohort studies have reported PFOS concentrations in blood and autopsy tissue data with PFOS present in sensitive organs across all human lifespan. However, to interpret such biomonitoring data in the context of chemical risk assessment, it is necessary to have a mechanistic framework that explains show the physiological changes across age affects the concentration of chemical inside different tissues of the human body. PBPK model is widely and successfully used in the field of risk assessment. The objective of this manuscript is to develop a dynamic age-dependent PBPK model as an extension of the previously published adult PFOS model and utilize this model to predict and compare the PFOS tissue distribution and plasma concentration across different age groups. Different cohort study data were used for exposure dose reconstruction and evaluation of time-dependent concentration in sensitive organs. Predicted plasma concentration followed trends observed in biomonitoring data and model predictions showed the increased disposition of PFOS in the geriatric population. PFOS model is sensitive to parameters governing renal resorption and elimination across all ages, which is related to PFOS half-life in humans. This model provides an effective framework for improving the quantitative risk assessment of PFOS throughout the human lifetime, particularly in susceptible age groups. The dynamic age-dependent PBPK model provides a step forward for developing such kind of dynamic model for other perfluoroalkyl substances.

Neurologic Manifestations of COVID-19 in Children: Emerging Pathophysiologic Insights.

The COVID-19 pandemic has affected mortality and morbidity across all ages, including children. Neurologic manifestations of coronavirus disease 2019, ranging from headaches to cerebrovascular stroke, may involve the CNS or peripheral nervous system. Neurologic involvement is also noted in the multisystem inflammatory syndrome in children, a pediatric condition that occurs weeks after infection with severe acute respiratory syndrome coronavirus 2, the virus that causes COVID-19. Knowledge about mechanisms of neurologic disease and immunologic response to severe acute respiratory syndrome coronavirus 2 is scarce but rapidly growing. Here, we concisely review experimental and clinical data relevant to pathophysiologic hypotheses regarding neurologic manifestations of coronavirus disease 2019. We highlight important knowledge gaps in differences between adults and children in their response to coronavirus disease 2019.

SARS-CoV-2 infection drives endothelial dysfunction indirectly through hypoxia and increased pro-inflammatory cytokine production

Abstract In the Covid-19 patients, vascular thrombosis is observed in the heart, lung, and other organs, leading to multiorgan morbidity and mortality. We obtained heart and lung tissues from autopsies of the patients who died from Covid-19 or unrelated causes as a control. We found that endothelial cells of the hearts and lungs were in dysfunctional state during Covid-19. Cardiac and pulmonary endothelial cells in the Covid-19 group showed decreased level of thrombomodulin, an anticoagulant, and increased level of von Willebrand factor, a procoagulant, compared to non-Covid-19 control. Endothelial cells in the Covid-19 autopsy hearts and lungs also exhibited reduced ICAM-1, VCAM-1, and E-selectin expression. Most of the hearts from the Covid-19 autopsies tested negative for SARS-CoV-2 genome, while all the lungs tested positive. Human coronary artery endothelial cell (HCAEC) in vitro culture was not infected by SARS-CoV-2, showing no dysfunctional phenotype. In the HCAEC culture and in vivo mouse experiments, hypoxia and pro-inflammatory cytokine treatment caused endothelial cell dysfunction including thrombomodulin downregulation similar to what we observed in Covid-19 autopsy tissues. Collectively, endothelial cell dysfunction associated with thrombus formation in Covid-19 is not caused by direct infection with SARS-CoV-2, but it can be induced by pathogenic microenvironments such as hypoxia and increased pro-inflammatory cytokine milieu.

COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

COVID-19,which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samplesfrom donors who died ofCOVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

Juxtacortical susceptibility changes in progressive multifocal leukoencephalopathy at the gray–white matter junction correlates with iron-enriched macrophages

Objective: Describe magnetic resonance imaging (MRI) susceptibility changes in progressive multifocal leukoencephalopathy (PML) and identify neuropathological correlates. Methods: PML cases and matched controls with primary central nervous system lymphoma (PCNSL) were retrospectively identified. MRI brain at 3 T and 7 T were reviewed. MRI-pathology correlations in fixed brain autopsy tissue were conducted in three subjects with confirmed PML. Results: With PML (n = 26 total, n = 5 multiple sclerosis natalizumab-associated), juxtacortical changes on susceptibility-weighted imaging (SWI) or gradient echo (GRE) sequences were noted in 3/3 cases on 7 T MRI and 14/22 cases (63.6%) on 1.5 T or 8/22 (36.4%) 3 T MRI. Similar findings were only noted in 3/25 (12.0%) of PCNSL patients (odds ratio (OR) 12.83, 95% confidence interval (CI), 2.9–56.7, p < 0.001) on 1.5 or 3 T MRI. On susceptibility sequences available prior to diagnosis of PML, 7 (87.5%) had changes present on average 2.7 ± 1.8 months (mean ± SD) prior to diagnosis. Postmortem 7 T MRI showed SWI changes corresponded to areas of increased iron density along the gray–white matter (GM-WM) junction predominantly in macrophages. Conclusion: Susceptibility changes in PML along the GM-WM junction can precede noticeable fluid-attenuated inversion recovery (FLAIR) changes and correlates with iron accumulation in macrophages.

Shotgun transcriptome, spatial omics, and isothermal profiling of SARS-CoV-2 infection reveals unique host responses, viral diversification, and drug interactions

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin–angiotensin–aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.

Thrombomodulin is upregulated in the kidneys of women with pre-eclampsia

The endothelial glycoprotein thrombomodulin regulates coagulation, vascular inflammation and apoptosis. In the kidney, thrombomodulin protects the glomerular filtration barrier by eliciting crosstalk between the glomerular endothelium and podocytes. Several glomerular pathologies are characterized by a loss of glomerular thrombomodulin. In women with pre-eclampsia, serum levels of soluble thrombomodulin are increased, possibly reflecting a loss from the glomerular endothelium. We set out to investigate whether thrombomodulin expression is decreased in the kidneys of women with pre-eclampsia and rats exposed to an angiogenesis inhibitor. Thrombomodulin expression was examined using immunohistochemistry and qPCR in renal autopsy tissues collected from 11 pre-eclamptic women, 22 pregnant controls and 11 hypertensive non-pregnant women. Further, kidneys from rats treated with increasing doses of sunitinib or sunitinib in combination with endothelin receptor antagonists were studied. Glomerular thrombomodulin protein levels were increased in the kidneys of women with pre-eclampsia. In parallel, in rats exposed to sunitinib, glomerular thrombomodulin was upregulated in a dose-dependent manner, and the upregulation of glomerular thrombomodulin preceded the onset of histopathological changes. Selective ETAR blockade, but not dual ETA/BR blockade, normalised the sunitinib-induced increase in thrombomodulin expression and albuminuria. We propose that glomerular thrombomodulin expression increases at an early stage of renal damage induced by antiangiogenic conditions. The upregulation of this nephroprotective protein in glomerular endothelial cells might serve as a mechanism to protect the glomerular filtration barrier in pre-eclampsia.

The Genetic Evolution of Treatment-Resistant Cutaneous, Acral, and Uveal Melanomas.

Melanoma is a biologically heterogeneous disease composed of distinct clinicopathologic subtypes that frequently resist treatment. To explore the evolution of treatment resistance and metastasis, we used a combination of temporal and multilesional tumor sampling in conjunction with whole-exome sequencing of 110 tumors collected from 7 patients with cutaneous (n = 3), uveal (n = 2), and acral (n = 2) melanoma subtypes. Primary tumors, metastases collected longitudinally, and autopsy tissues were interrogated. All but 1 patient died because of melanoma progression. For each patient, we generated phylogenies and quantified the extent of genetic diversity among tumors, specifically among putative somatic alterations affecting therapeutic resistance. In 4 patients who received immunotherapy, we found 1-3 putative acquired and intrinsic resistance mechanisms coexisting in the same patient, including mechanisms that were shared by all tumors within each patient, suggesting that future therapies directed at overcoming intrinsic resistance mechanisms may be broadly effective.

Borrelia Invasion of Brain Pyramidal Neurons and Biofilm Borrelia Plaques in Neuroborreliosis Dementia with Alzheimer’s Phenotype

Dementia in Lyme borreliosis complex has been reported, mainly in post-mortem studies without available antemortem evidence of active borrelia infection. Blanc in 2014 studied living patients with Lyme neuroborreliosis dementia and several dementia phenotype illnesses including an Alzheimer’s Phenotype. Herein we report an additional case study of a longitudinal evolution of European neuroborreliosis over eight years from tick bite to mild cognitive disease, to advanced dementia to death with a brain Alzheimer’s disease phenotype and concurrent borrelia deposits in brain Alzheimer’s disease sites at autopsy. Intrathecal borrelia specific antibodies were detected by commercial diagnostic laboratories (antemortem). Molecular autopsy tissue imaging was completed with borrelia specific DNA probes and an immunomicroscopic detection histopathology method. Results: Autopsy showed intact spirochetes, fragmented spirochetes, deposits of borrelia-specific proteins inside plaque lesions and inside of neurons, and borrelia DNA deposits in plaque and neuronal sites. Pure Alzheimer’s disease (without Lewy bodies) was a routine neuropathological finding. CSF evidence for a brain compartment immune response is established here. Intrathecal antibodies to infection presented as oligoclonal total CSF IgG bands (n=twelve increase to n=13 bands) and separate borrelia IgG western blot band analysis in cerebrospinal fluids (seven diagnostic borrelia CSF antibody bands). Blood western blot disclosed triple borrelia species infection; burgdorferi European type (eighteen bands), garinii (twelve bands) and afzelii (eighteen bands). Total borrelia IgG antibodies in blood during life were two hundred-fold higher than normal range. Western blot of cerebrospinal fluid prior to death disclosed 7 protein bands which were not represented in simultaneous blood western blot studies, further validating the intrathecal fingerprint of a separate brain compartment immune response to neuroborreliosis infection. Conclusion: Borrelia protein antigenic stimulation of intrathecal borrelia antibodies was caused by resident deposits of spirochetal protein deposits in plaques, in diseased neurons, and in neuropil brain sites, and in intact brain spirochetes. Deposits of borrelia proteins inside neurons and brain phagocytes and in neuropil sites (invasosomes) confirm remnants of chronic brain infection.