LT and SOX9 expression are associated with gene sets that distinguish Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive malignant neuroendocrine tumour. There are two subsets of MCC, one related to Merkel cell polyomavirus (MCPyV) and the other to ultraviolet radiation (UVR). MCPyV-positive and MCPyV-negative MCCs have been considered to be different tumours, as the former harbour few DNA mutations and are not related to UVR, and the latter usually arise in sun-exposed areas and may be found in conjunction with other keratinocytic tumours, mostly squamous cell carcinomas. Two viral oncoproteins, large T antigen (LT; coded by MCPyV_gp3) and small T antigen (sT; coded by MCPyV_gp4), promote different carcinogenic pathways. OBJECTIVES: To determine which genes are differentially expressed in MCPyV-positive and MCPyV-negative MCC; to describe the mutational burden and the most frequently mutated genes in both MCC subtypes; and to identify the clinical and molecular factors that may be related to patient survival. METHODS: Ninety-two patients with a diagnosis of MCC were identified from the medical databases of participating centres. To study gene expression, a customized panel of 172 genes was developed. Gene expression profiling was performed with nCounter technology. For mutational studies, a customized panel of 26 genes was designed. Somatic single nucleotide variants (SNVs) were identified following the GATK Best Practices workflow for somatic mutations. RESULTS: The expression of LT enabled the series to be divided into two groups (LT positive, n = 55; LT negative, n = 37). Genes differentially expressed in LT-negative patients were related to epithelial differentiation, especially SOX9, or proliferation and the cell cycle (MYC, CDK6), among others. Congruently, LT displayed lower expression in SOX9-positive patients, and differentially expressed genes in SOX9-positive patients were related to epithelial/squamous differentiation. In LT-positive patients, the mean SNV frequency was 4.3; in LT-negative patients it was 10 (P = 0.03). On multivariate survival analysis, the expression of SNAI1 [hazard ratio (HR) 1.046, 95% confidence interval (CI) 1.007-1.086; P = 0.02] and CDK6 (HR 1.049, 95% CI 1.020-1.080; P = 0.001) were identified as risk factors. CONCLUSIONS: Tumours with weak LT expression tend to co-express genes related to squamous differentiation and the cell cycle, and to have a higher mutational burden. These findings are congruent with those of earlier studies.

Merkel cell carcinoma (MCC) is an aggressive form of skin tumour. There are two subtypes of MCC: one of them is related to a virus called Merkel cell polyomavirus (MCPyV); the other one is related to persistent exposure to sunlight. The aim of this research was to find differences between these subtypes in their molecular behaviour (the genes that are expressed and the mutations that may be found). To do this, we carried out two studies, one to investigate gene expression (the process cells use to convert the instructions in our DNA into a functional product such as a protein) and one to look at gene mutations (changes in the DNA sequence). We found that the tumours that were not related to MCPyV expressed genes related to epithelial differentiation (the process by which unspecialized cells gain features characteristics of epithelial cells, which, among other things, make up the outer surface of the body), which means that the origin of both MCC subtypes may be different. We also found that MCPyV-related tumours had fewer mutations. Our findings are important because they help us to understand the biology of the MCC subtypes and could help with the development of new treatments for people diagnosed with skin tumours.

Immune and stromal transcriptional patterns that influence the outcome of classic Hodgkin lymphoma.

Classic Hodgkin lymphoma (cHL) is characterized by a rich immune microenvironment as the main tumor component. It involves a broad range of cell populations, which are largely unexplored, even though they are known to be essential for growth and survival of Hodgkin and Reed-Sternberg cells. We profiled the gene expression of 25 FFPE cHL samples using NanoString technology and resolved their microenvironment compositions using cell-deconvolution tools, thereby generating patient-specific signatures. The results confirm individual immune fingerprints and recognize multiple clusters enriched in refractory patients, highlighting the relevance of: (1) the composition of immune cells and their functional status, including myeloid cell populations (M1-like, M2-like, plasmacytoid dendritic cells, myeloid-derived suppressor cells, etc.), CD4-positive T cells (exhausted, regulatory, Th17, etc.), cytotoxic CD8 T and natural killer cells; (2) the balance between inflammatory signatures (such as IL6, TNF, IFN-γ/TGF-ß) and MHC-I/MHC-II molecules; and (3) several cells, pathways and genes related to the stroma and extracellular matrix remodeling. A validation model combining relevant immune and stromal signatures identifies patients with unfavorable outcomes, producing the same results in an independent cHL series. Our results reveal the heterogeneity of immune responses among patients, confirm previous findings, and identify new functional phenotypes of prognostic and predictive utility.

Extranodal natural killer/T-cell lymphoma nasal type in a western population: Molecular profiling identifies new therapeutic targets.

(A) Correlation matrix of unsupervised co-regulated genes, based on the 208 genes included in the NanoString platform. Some of the clusters of co-regulated genes corresponded to the following: Inflammatory cells; Epstein-Barr virus; B-cells; Cytotoxic T-cells; T-cells; and Proliferation. (B) Analysis of genomic alterations by targeted sequencing. Distribution of mutations in the 62 analyzed genes. Rows correspond to sequenced genes, columns represent individual patients. Color coding: green, missense; blue, synonymous; pink, frameshift; violet, Indel; red, stop gained; yellow, UTR.

HPV infection profile in cervical lesions.

INTRODUCTION: The prevalence of the different genotypes of human papillomavirus (HPV) varies depending on lesion severity and geographic region. OBJECTIVE: To identify multiple HPV infections in low- and high-grade cervical lesions in a group of women from the Mexican Bajío region referred with inconclusive cytology. METHODS: Pilot study of women referred from primary care units of Guanajuato, Mexico, with cytology suggestive of cervical lesion. Cervical smears were subjected to DNA extraction and HPV genotyping using microarrays. RESULTS: 100 consecutive cases were collected and 90 were analyzed; HPV positivity was observed in 26% of healthy women, and 62% had some degree of cervical lesion. The most common HPV genotypes were 59, 31, 16 and 51. Multiple infections were found in most samples. CONCLUSIONS: HPV heterogeneity was identified in the samples of the study population in contrast to worldwide reports; furthermore, multiple infections are common in precursor lesions and decrease in high-grade lesions. These data could have an impact on current HPV vaccination programs.

INTRODUCCIÓN: La prevalencia de los diferentes genotipos de virus del papiloma humano (VPH) varía dependiendo de la severidad de la lesión y región geográfica. OBJETIVO: Identificar infecciones múltiples de VPH en lesiones cervicales de bajo y alto grado en un grupo de mujeres del Bajío mexicano referidas con citología no concluyente. MÉTODOS: Estudio piloto de mujeres referidas de unidades del primer nivel de atención de Guanajuato, México, por citología sugerente de lesión cervical. Los raspados cervicales fueron sujetos a extracción de ADN y genotipificación del VPH mediante microarreglos. RESULTADOS: Se colectaron 100 casos consecutivos y fueron analizados 90; se observó 26 % de positividad a VPH en mujeres sanas y 62 % presentó algún grado de lesión. Los genotipos de VPH más frecuentes fueron 59, 31, 16 y 51. En la mayoría de las muestras se encontró infección múltiple. CONCLUSIONES: Se identificó heterogeneidad de VPH en las muestras de la población estudiada en contraste con los reportes internacionales; además, son comunes las infecciones múltiples en lesiones precursoras y disminuyen en las lesiones de alto grado. Estos datos podrían influir en los actuales programas de vacunación anti-VPH.

An integrated prognostic model for diffuse large B-cell lymphoma treated with immunochemotherapy.

Diffuse large B-cell lymphoma (DLBCL), the most frequent non-Hodgkin's lymphoma subtype, is characterized by strong biological, morphological, and clinical heterogeneity, but patients are treated with immunochemotherapy in a relatively homogeneous way. Here, we have used a customized NanoString platform to analyze a series of 197 homogeneously treated DLBCL cases. The platform includes the most relevant genes or signatures known to be useful for predicting response to R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) in DLBCL cases. We generated a risk score that combines the International Prognostic Index with cell of origin and double expression of MYC/BCL2, and stratified the series into three groups, yielding hazard ratios from 0.15 to 5.49 for overall survival, and from 0.17 to 5.04 for progression-free survival. Group differences were highly significant (p < 0.0001), and the scoring system was applicable to younger patients (<60 years of age) and patients with advanced or localized stages of the disease. Results were validated in an independent dataset from 166 DLBCL patients treated in two distinct clinical trials. This risk score combines clinical and biological data in a model that can be used to integrate biological variables into the prognostic models for DLBCL cases.

Perfil de infecciones por VPH en lesiones cervicales / HPV infection profile in cervical lesions

Resumen Introducción: La prevalencia de los diferentes genotipos de virus del papiloma humano (VPH) varía dependiendo de la severidad de la lesión y región geográfica. Objetivo: Identificar infecciones múltiples de VPH en lesiones cervicales de bajo y alto grado en un grupo de mujeres del Bajío mexicano referidas con citología no concluyente. Métodos: Estudio piloto de mujeres referidas de unidades del primer nivel de atención de Guanajuato, México, por citología sugerente de lesión cervical. Los raspados cervicales fueron sujetos a extracción de ADN y genotipificación del VPH mediante microarreglos. Resultados: Se colectaron 100 casos consecutivos y fueron analizados 90; se observó 26 % de positividad a VPH en mujeres sanas y 62 % presentó algún grado de lesión. Los genotipos de VPH más frecuentes fueron 59, 31, 16 y 51. En la mayoría de las muestras se encontró infección múltiple. Conclusiones: Se identificó heterogeneidad de VPH en las muestras de la población estudiada en contraste con los reportes internacionales; además, son comunes las infecciones múltiples en lesiones precursoras y disminuyen en las lesiones de alto grado. Estos datos podrían influir en los actuales programas de vacunación anti-VPH.

Abstract Introduction: The prevalence of the different genotypes of human papillomavirus (HPV) varies depending on lesion severity and geographic region Objective: To identify multiple HPV infections in low- and high-grade cervical lesions in a group of women from the Mexican Bajío region referred with inconclusive cytology. Methods: Pilot study of women referred from primary care units of Guanajuato, Mexico, with cytology suggestive of cervical lesion. Cervical smears were subjected to DNA extraction and HPV genotyping using microarrays. Results: 100 consecutive cases were collected and 90 were analyzed; HPV positivity was observed in 26% of healthy women, 62% had some degree of cervical lesion. The most common HPV genotypes were 59, 31, 16 and 51. Multiple infections were found in most samples. Conclusions: HPV heterogeneity was identified in the samples of the study population in contrast to worldwide reports; furthermore, multiple infections are common in precursor lesions and decrease in high-grade lesions. These data could have an impact on current HPV vaccination programs.

PLCγ1/PKCθ Downstream Signaling Controls Cutaneous T-Cell Lymphoma Development and Progression.

Developing mechanistic rationales can improve the clinical management of cutaneous T-cell lymphomas. There is considerable genetic and biological evidence of a malignant network of signaling mechanisms, highly influenced by deregulated TCR/PLCγ1 activity, controlling the biology of these lesions. In addition, activated signal transducer and activator of transcription 3 is associated with clinical progression, although the alterations responsible for this have not been fully elucidated. Here, we studied PLCγ1-dependent mechanisms that can mediate STAT3 activation and control tumor growth and progression. Downstream of PLCγ1, the pharmacological inhibition and genetic knockdown of protein kinase C theta (PKCθ) inhibited signal transducer and activator of transcription 3 activation, impaired proliferation, and promoted apoptosis in cutaneous T-cell lymphoma cells. A PKCθ-dependent transcriptome in mycosis fungoides/Sézary syndrome cells revealed potential effector genes controlling cytokine signaling, TP53, and actin cytoskeleton dynamics. Consistently, an in vivo chicken embryo model xenografted with mycosis fungoides cells showed that PKCθ blockage abrogates tumor growth and spread to distant organs. Finally, the expression of a number of PKCθ target genes found in mycosis fungoides cells significantly correlated with that of PRKCQ (PKCθ) in 81 human mycosis fungoides samples. In summary, PKCθ can play a central role in the activation of malignant cutaneous T-cell lymphoma mechanisms via multiple routes, including, but not restricted to, STAT3. These mechanisms may, in turn, serve as targets for specific therapies.

Peripheral T-cell lymphoma: molecular profiling recognizes subclasses and identifies prognostic markers.

Peripheral T-cell lymphoma (PTCL) is a clinically aggressive disease, with a poor response to therapy and a low overall survival rate of approximately 30% after 5 years. We have analyzed a series of 105 cases with a diagnosis of PTCL using a customized NanoString platform (NanoString Technologies, Seattle, WA) that includes 208 genes associated with T-cell differentiation, oncogenes and tumor suppressor genes, deregulated pathways, and stromal cell subpopulations. A comparative analysis of the various histological types of PTCL (angioimmunoblastic T-cell lymphoma [AITL]; PTCL with T follicular helper [TFH] phenotype; PTCL not otherwise specified [NOS]) showed that specific sets of genes were associated with each of the diagnoses. These included TFH markers, cytotoxic markers, and genes whose expression was a surrogate for specific cellular subpopulations, including follicular dendritic cells, mast cells, and genes belonging to precise survival (NF-κB) and other pathways. Furthermore, the mutational profile was analyzed using a custom panel that targeted 62 genes in 76 cases distributed in AITL, PTCL-TFH, and PTCL-NOS. The main differences among the 3 nodal PTCL classes involved the RHOAG17V mutations (P < .0001), which were approximately twice as frequent in AITL (34.09%) as in PTCL-TFH (16.66%) cases but were not detected in PTCL-NOS. A multivariate analysis identified gene sets that allowed the series of cases to be stratified into different risk groups. This study supports and validates the current division of PTCL into these 3 categories, identifies sets of markers that can be used for a more precise diagnosis, and recognizes the expression of B-cell genes as an IPI-independent prognostic factor for AITL.

Subcutaneous panniculitis-like T-cell lymphoma, lupus erythematosus profundus, and overlapping cases: molecular characterization through the study of 208 genes.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare cytotoxic cutaneous lymphoma. Differential diagnosis with lupus erythematosus panniculitis (LEP) can be challenging and overlapping cases have been described. In this study, we investigate whether gene expression profiling may or not identify markers that can be used to improve our understanding of the disease and to make a precise differential diagnosis. SPTCL, LEP, and overlapping cases were analyzed using a customized NanoString platform including 208 genes related to T-cell differentiation, stromal signatures, oncogenes, and tumor suppressor genes. Gene expression unsupervised analysis of the samples differentiated SPTCL from LEP samples. Most overlapping cases were clustered with LEP cases. Differentially expressed genes were observed when comparing SPTCL with LEP cases; and overlapping with LEP cases. Gene set enrichment analysis recognized gene sets defining each group. In conclusion, SPTCL and LEP have distinctive molecular profiles and the molecular background of overlapping cases more closely resembles LEP.

An analysis of genetic targets for guiding clinical management of follicular lymphoma.

Introduction: Follicular lymphoma (FL) is one of the most common non-Hodgkin lymphoma (NHL) types, where genomic studies have accumulated potentially useful information about frequently mutated genes and deregulated pathways, which has allowed to a better understanding of the molecular pathogenesis of this tumor and the complex interrelationship between the tumoral cells and the stroma. Areas covered: The results of the molecular studies performed on Follicular Lymphoma have been here reviewed, summarizing the results of the clinical trials so far developed on this basis and discussing the reasons for the successes and failures. Searches were performed on June 1st, 2020, in PubMed and ClinicalTrials.gov. Expert opinion: Targeted therapy for follicular lymphoma has multiple opportunities including the use of epigenetic drugs, PI3K inhibitors, modifiers of the immune stroma and others. Data currently known on FL pathogenesis suggest that combining these treatments with immunotherapy should be explored in clinical trials, mainly for patients with clinical progression or adverse prognostic markers. Association of targeted trials with dynamic molecular studies of the tumor and serum samples is advised. Chemotherapy-free approaches should also be explored as first-line therapy for FL patients.

MicroRNA-199b Deregulation Shows a Strong SET-Independent Prognostic Value in Early-Stage Colorectal Cancer.

The endogenous PP2A inhibitor SET Nuclear Proto-Oncogene (SET) has been reported to play oncogenic roles and determines poor outcomes in colorectal cancer (CRC). Our group previously showed that miR-199b is deregulated in metastatic CRC, and reduced the cell viability and enhanced the sensitivity of CRC cells to standard induction chemotherapy drugs, mainly through direct negative SET regulation. Clinically, miR-199b downregulation was identified as the molecular mechanism responsible for SET overexpression in around half of metastatic CRC patients. However, the potential clinical value of miR-199b in early-stage CRC remains totally unknown. Thus, here we explored the expression levels of this microRNA in a cohort of 171 early-stage CRC patients using real-time polymerase chain reactions. MiR-199b downregulation was found in 21.6% of cases (37 out of 171) and was significantly associated with those patients with a worse Eastern Cooperative Oncology Group (ECOG) status (p = 0.045). Moreover, miR-199b downregulation predicted shorter overall (p < 0.001) and progression-free survival (p = 0.015). As expected, we next immunohistochemically analyzed SET, observing that it was significantly associated with miR-199b in our cohort. However, multivariate analyses showed that miR-199b was an independent biomarker of poor outcomes in early-stage CRC with a predictive value stronger than SET. In conclusion, our results highlight the potential clinical usefulness of miR-199b and suggest that it could represent a novel molecular target in this disease.

Identification of tipifarnib sensitivity biomarkers in T-cell acute lymphoblastic leukemia and T-cell lymphoma.

Patients diagnosed with T-cell leukemias and T-cell lymphomas (TCLs) still have a poor prognosis and an inadequate response to current therapies, highlighting the need for targeted treatments. We have analyzed the potential therapeutic value of the farnesyltransferase inhibitor, tipifarnib, in 25 TCL cell lines through the identification of genomic and/or immunohistochemical markers of tipifarnib sensitivity. More than half of the cell lines (60%) were considered to be sensitive. Tipifarnib reduced cell viability in these T-cell leukemia and TCL cell lines, induced apoptosis and modified the cell cycle. A mutational study showed TP53, NOTCH1 and DNMT3 to be mutated in 84.6%, 69.2% and 30.0% of sensitive cell lines, and in 62.5%, 0% and 0% of resistant cell lines, respectively. An immunohistochemistry study showed that p-ERK and RelB were associated as potential biomarkers of tipifarnib sensitivity and resistance, respectively. Data from RNA-seq show that tipifarnib at IC50 after 72 h downregulated a great variety of pathways, including those controlling cell cycle, metabolism, and ribosomal and mitochondrial activity. This study establishes tipifarnib as a potential therapeutic option in T-cell leukemia and TCL. The mutational state of NOTCH1, p-ERK and RelB could serve as potential biomarkers of tipifarnib sensitivity and resistance.

Parkinson's Disease: A Comprehensive Analysis of Fungi and Bacteria in Brain Tissue.

Parkinson's disease (PD) is characterized by motor disorders and the destruction of dopaminergic neurons in the substantia nigra pars compacta. In addition to motor disability, many patients with PD present a spectrum of clinical symptoms, including cognitive decline, psychiatric alterations, loss of smell and bladder dysfunction, among others. Neuroinflammation is one of the most salient features of PD, but the nature of the trigger remains unknown. A plausible mechanism to explain inflammation and the range of clinical symptoms in these patients is the presence of systemic microbial infection. Accordingly, the present study provides extensive evidence for the existence of mixed microbial infections in the central nervous system (CNS) of patients with PD. Assessment of CNS sections by immunohistochemistry using specific antibodies revealed the presence of both fungi and bacteria. Moreover, different regions of the CNS were positive for a variety of microbial morphologies, suggesting infection by a number of microorganisms. Identification of specific fungal and bacterial species in different CNS regions from six PD patients was accomplished using nested PCR analysis and next-generation sequencing, providing compelling evidence of polymicrobial infections in the CNS of PD. Most of the fungal species identified belong to the genera Botrytis, Candida, Fusarium and Malassezia. Some relevant bacterial genera were Streptococcus and Pseudomonas, with most bacterial species belonging to the phyla Actinobacteria and Proteobacteria. Interestingly, we noted similarities and differences between the microbiota present in the CNS of patients with PD and that in other neurodegenerative diseases. Overall, our observations lend strong support to the concept that mixed microbial infections contribute to or are a risk factor for the neuropathology of PD. Importantly, these results provide the basis for effective treatments of this disease using already approved and safe antimicrobial therapeutics.

Update on peripheral T-cell lymphomas with T-helper phenotype: Are there too many subtypes?

Follicular helper T (TFH) cells are the providers of T-cell help to B-cells in the development of germinal centers and for the generation of most class-switched antibodies. The markers most commonly associated with TFH activity are IL21, IL4, CD40L, BCL6, SAP, CXCR5/CXCL13, and ICOS. T-cell lymphoma genomic studies have shown that different T-cell lymphoma types express signatures typical for TFH cells, this including angioimmunoblastic T-cell lymphoma (AITL), a related condition termed peripheral T-cell lymphoma with TFH phenotype and primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder. Angioimmunoblastic T-cell lymphoma is a well-established entity, a clinically aggressive disease with a survival of 30% OS after 5 years. Molecular and clinical studies have confirmed this as a well-established clinicopathological entity with relatively specific gene mutations, including mutations found in hematopoietic precursor cells and others. Peripheral T-cell lymphoma with TFH phenotype is an associated disorder with histology of PTCL but a TFH phenotype, as defined by the expression of 2-3 immunohistochemical markers. Molecular studies on this entity are showing a partial overlap with AITL. Primary cutaneous CD4+ small/medium lymphoproliferative disorder is an entirely different process that takes place in the skin, showing frank cytologic atypia, monoclonal TCR rearrangement and TFH phenotype in the context of a clinically benign lesion. Here we review the main clinical, molecular and diagnostic features of these three lymphoproliferative processes.

Brain Microbiota in Huntington's Disease Patients.

One of the most important challenges facing medical science is to better understand the cause of neuronal pathology in neurodegenerative diseases. Such is the case for Huntington's disease (HD), a genetic disorder primarily caused by a triplet expansion in the Huntingtin gene (HTT). Although aberrant HTT is expressed from embryogenesis, it remains puzzling as to why the onset of disease symptoms manifest only after several decades of life. In the present study, we investigated the possibility of microbial infection in brain tissue from patients with HD, reasoning that perhaps mutated HTT could be deleterious for immune cells and neural tissue, and could facilitate microbial colonization. Using immunohistochemistry approaches, we observed a variety of fungal structures in the striatum and frontal cortex of seven HD patients. Some of these fungi were found in close proximity to the nucleus, or even as intranuclear inclusions. Identification of the fungal species was accomplished by next-generation sequencing (NGS). Interestingly, some genera, such as Ramularia, appeared unique to HD patients, and have not been previously described in other neurodegenerative diseases. Several bacterial species were also identified both by PCR and NGS. Notably, a curved and filamentous structure that immunoreacts with anti-bacterial antibodies was characteristic of HD brains and has not been previously observed in brain tissue from neurodegenerative patients. Prevalent bacterial genera included Pseudomonas, Acinetobacter, and Burkholderia. Collectively, our results represent the first attempt to identify the brain microbiota in HD. Our observations suggest that microbial colonization may be a risk factor for HD and might explain why the onset of the disease appears after several decades of life. Importantly, they may open a new field of investigation and could help in the design of new therapeutic strategies for this devastating disorder.

Searching for Bacteria in Neural Tissue From Amyotrophic Lateral Sclerosis.

Despite great efforts in the investigation, the exact etiology of amyotrophic lateral sclerosis (ALS) is a matter of intensive research. We recently advanced the idea that ALS might be caused by fungal infection. Indeed, fungal yeast and hyphal structures can be directly visualized in neural tissue of ALS patients, and a number of fungal species have been identified in the central nervous system (CNS). In the present work, we tested the possibility that bacterial infections can accompany these mycoses. Our findings establish the presence of bacterial DNA in different regions of the CNS from all ALS patients examined. Specifically, we used PCR and next generation sequencing (NGS) to precisely determine the bacterial species present in ALS tissue. Consistent with these findings, immunohistochemistry analysis of CNS sections using specific anti-bacterial antibodies identified prokaryotic cells in neural tissue. Finally, we assayed for the repeat expansion of the hexanucleotide repeat GGGGCC in C9orf72, which is considered the most common genetic cause of ALS in patients, using DNA extracted from ALS CNS tissue. We failed to find this repeated sequence in any of the eleven patients analyzed. Our results indicate that bacterial DNA and prokaryotic cells are present in CNS tissue, leading to the concept that both fungal and bacterial infections coexist in patients with ALS. These observations lay the groundwork for the use of appropriate therapies to eradicate the polymicrobial infections in ALS.

Human and Microbial Proteins From Corpora Amylacea of Alzheimer's Disease.

Corpora amylacea (CA) are spherical bodies mainly composed of polyglucans and, to a lesser extent, proteins. They are abundant in brains from patients with neurodegenerative diseases, particularly Alzheimer's disease. Although CA were discovered many years ago, their precise origin and function remain obscure. CA from the insular cortex of two Alzheimer's patients were purified and the protein composition was assessed by proteomic analysis. A number of microbial proteins were identified and fungal DNA was detected by nested PCR.A wide variety of human proteins form part of CA. In addition, we unequivocally demonstrated several fungal and bacterial proteins in purified CA. In addition to a variety of human proteins, CA also contain fungal and bacterial polypeptides.In conclusion, this paper suggests that the function of CA is to scavenge cellular debris provoked by microbial infections.

Infection of Fungi and Bacteria in Brain Tissue From Elderly Persons and Patients With Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of dementia in elderly people. The etiology of this disease remains a matter of intensive research in many laboratories. We have advanced the idea that disseminated fungal infection contributes to the etiology of AD. Thus, we have demonstrated that fungal proteins and DNA are present in nervous tissue from AD patients. More recently, we have reported that bacterial infections can accompany these mycoses, suggesting that polymicrobial infections exist in AD brains. In the present study, we have examined fungal and bacterial infection in brain tissue from AD patients and control subjects by immunohistochemistry. In addition, we have documented the fungal and bacterial species in brain regions from AD patients and control subjects by next-generation sequencing (NGS). Our results from the analysis of ten AD patients reveal a variety of fungal and bacterial species, although some were more prominent than others. The fungal genera more prevalent in AD patients were Alternaria, Botrytis, Candida, and Malassezia. We also compared these genera with those found in elderly and younger subjects. One of the most prominent genera in control subjects was Fusarium. Principal component analysis clearly indicated that fungi from frontal cortex samples of AD brains clustered together and differed from those of equivalent control subjects. Regarding bacterial infection, the phylum Proteobacteria was the most prominent in both AD patients and controls, followed by Firmicutes, Actinobacteria, and Bacteroides. At the family level, Burkholderiaceae and Staphylococcaceae exhibited higher percentages in AD brains than in control brains. These findings could be of interest to guide targeted antimicrobial therapy for AD patients. Moreover, the variety of microbial species in each patient may constitute a basis for a better understanding of the evolution and severity of clinical symptoms in each patient.

Multiple sclerosis and mixed microbial infections. Direct identification of fungi and bacteria in nervous tissue.

Multiple sclerosis (MS) is the prototypical inflammatory disease of the central nervous system (CNS), leading to multifocal demyelination and neurodegeneration. The etiology of this incurable disease is unknown and remains a matter of intensive research. The possibility that microbial infections, such as viruses or bacteria, can trigger an autoimmune reaction in CNS tissue has been suggested. However, the recent demonstration that bacteria are present in CNS tissue points to a direct involvement of microbial infections in the etiology of MS. In the present study, we provide the first evidence of fungal infection in CNS tissue of MS patients, and demonstrate that fungal DNA from different species can be detected in the CNS. We used, nested PCR assays together with next-generation sequencing to identify the fungal species in the nervous tissue of 10 patients with MS. Strikingly, Trichosporon mucoides was found in the majority of MS patients, and particularly high levels of this fungus were found in two patients. Importantly, T. mucoides was not detected in the CNS of control subjects. We were also able to visualize fungal structures in CNS tissue sections by immunohistochemistry using specific antifungal antibodies, which also revealed the accumulation of a number of microbial cells in microfoci. Again, microbial structures were not observed in CNS sections from controls. In addition to fungi, neural tissue from MS patients was also positive for bacteria. In conclusion, our present observations point to the novel concept that MS could be caused by polymicrobial infections. Thus, mycosis of the CNS may be accompanied by opportunistic bacterial infection, promoting neuroinflammation and directly causing focal lesions, followed by demyelination and axonal injury.