Diana Pisa

Different Brain Regions are Infected with Fungi in Alzheimer's Disease.

The possibility that Alzheimer’s disease (AD) has a microbial aetiology has been proposed by several researchers. Here, we provide evidence that tissue from the central nervous system (CNS) of AD patients contain fungal cells and hyphae. Fungal material can be detected both intra- and extracellularly using specific antibodies against several fungi. Different brain regions including external frontal cortex, cerebellar hemisphere, entorhinal cortex/hippocampus and choroid plexus contain fungal material, which is absent in brain tissue from control individuals. Analysis of brain sections from ten additional AD patients reveals that all are infected with fungi. Fungal infection is also observed in blood vessels, which may explain the vascular pathology frequently detected in AD patients. Sequencing of fungal DNA extracted from frozen CNS samples identifies several fungal species. Collectively, our findings provide compelling evidence for the existence of fungal infection in the CNS from AD patients, but not in control individuals.

Fungal infection in patients with Alzheimer's disease.

Alzheimers disease is a progressive neurodegenerative disorder that leads to dementia mainly among the elderly. This disease is characterized by the presence in the brain of amyloid plaques and neurofibrillary tangles that provoke neuronal cell death, vascular dysfunction, and inflammatory processes. In the present work, we have analyzed the existence of fungal infection in Alzheimers disease patients. A proteomic analysis provides compelling evidence for the existence of fungal proteins in brain samples from Alzheimers disease patients. Furthermore, PCR analysis reveals a variety of fungal species in these samples, dependent on the patient and the tissue tested. DNA sequencing demonstrated that several fungal species can be found in brain samples. Together, these results show that fungal macromolecules can be detected in brain from Alzheimers disease patients. To our knowledge these findings represent the first evidence that fungal infection is detectable in brain samples from Alzheimers disease patients. The possibility that this may represent a risk factor or may contribute to the etiological cause of Alzheimers disease is discussed.

Isolation of Candida famata from a Patient with Acute Zonal Occult Outer Retinopathy

ABSTRACT The etiology of a number of retinopathies, such as acute zonal occult outer retinopathy (AZOOR), remains undetermined. Candida famata was isolated from conjunctival exudates of a patient diagnosed with AZOOR. This yeast was very abundant, particularly in the more affected eye, while no other pathogens or fungal species were in evidence. Immunological tests revealed the presence of antigen-specific T lymphocytes by using C. famata as a challenge. Moreover, enzyme-linked immunosorbent assay analysis showed the presence of specific antibodies against this yeast in the patients blood. Delayed hypersensitivity by use of a skin test was also positive. Finally, antifungal treatments led to improvements in several clinical symptoms, including funduscopic analysis. However, despite prolonged treatment with fluconazole and itraconazole, C. famata still appeared in the conjunctival exudates. The new antifungal voriconazole may represent a better choice for treatment.

Vitamin D3 Receptor (VDR) Gene rs2228570 (Fok1) and rs731236 (Taq1) Variants Are Not Associated with the Risk for Multiple Sclerosis: Results of a New Study and a Meta-Analysis

Background Some epidemiological, genetic, and experimental data suggest a possible role of vitamin D in the pathogenesis of multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis. Data on the possible contribution of several single nucleotide polymorphisms (SNP) in the vitamin D receptor (VDR) gene to the risk for MS are controversial. Several studies suggested an interaction between some SNPs in the VDR gene and HLADRB1*1501 in the risk for MS. Objectives The aim of this study was to investigate a possible influence of the SNPs rs2228570 and rs731236 in the VDR gene in the risk for MS. A secondary objective was to address the possible interactions between VDR genes and HLADRB1*1501. Methods We analyzed the allelic and genotype frequency of VDR rs2228570, rs731236, and HLADRB1*1501 (rs3135388) in 303 patients with MS and 310 healthy controls, using TaqMan Assays. We also conducted a meta-analysis, that was carried out by using the software Meta-Disc 1.1.1 (http://www.hrc.es/investigacion/metadisc.html; Unit of Clinical Statistics, Hospital Ramón y Cajal, Madrid, Spain). Heterogeneity between studies in terms of degree of association was tested using the Q-statistic. Results VDR rs2228570 and rs731236 allelic and genotype frequencies did not differ significantly between MS patients and controls, and were unrelated with the age of onset of MS, gender, and course of MS. HLADRB1*1501 showed a high association with the risk of developing MS 4.76(95% C.I.  = 3.14–7.27; p<0.0001). The meta-analysis, after excluding data of one study that was responsible of heterogeneity for rs731236 polymorphism, showed lack of relation of both SNPs with the risk for MS. HLADRB1*1501 showed lack of interaction with VDR rs2228570 and rs731236 in increasing MS risk. Conclusions These results suggest that VDR rs2228570 and rs731236 polymorphisms are not related with the risk for MS, and did not confirm interaction between these VDR SNPs and HLADRB1 in the risk for MS.

Direct Visualization of Fungal Infection in Brains from Patients with Alzheimer's Disease

Recently, we have reported the presence of fungal infections in patients with Alzheimers disease (AD). Accordingly, fungal proteins and DNA were found in brain samples, demonstrating the existence of infection in the central nervous system. In the present work, we raised antibodies to specific fungal species and performed immunohistochemistry to directly visualize fungal components inside neurons from AD patients. Mice infected with Candida glabrata were initially used to assess whether yeast can be internalized in mammalian tissues. Using polyclonal rabbit antibodies against C. glabrata, rounded immunopositive cells could be detected in the cytoplasm of cells from liver, spleen, and brain samples in infected, but not uninfected, mice. Immunohistochemical analyses of tissue from the frontal cortex of AD patients revealed the presence of fungal material in a small percentage (~10%) of cells, suggesting the presence of infection. Importantly, this immunopositive material was absent in control samples. Confocal microscopy indicated that this fungal material had an intracellular localization. The specific morphology of this material varied between patients; in some instances, disseminated material was localized to the cytoplasm, whereas small punctate bodies were detected in other patients. Interestingly, fungal material could be revealed using different anti-fungal antibodies, suggesting multiple infections. In summary, fungal infection can only be observed using specific anti-fungal antibodies and only a small percentage of cells contain fungi. Our findings provide an explanation for the hitherto elusive detection of fungi in AD brains, and are consistent with the idea that fungal cells are internalized inside neurons.

Evidence for fungal infection in cerebrospinal fluid and brain tissue from patients with amyotrophic lateral sclerosis

Among neurogenerative diseases, amyotrophic lateral sclerosis (ALS) is a fatal illness characterized by a progressive motor neuron dysfunction in the motor cortex, brainstem and spinal cord. ALS is the most common form of motor neuron disease; yet, to date, the exact etiology of ALS remains unknown. In the present work, we have explored the possibility of fungal infection in cerebrospinal fluid (CSF) and in brain tissue from ALS patients. Fungal antigens, as well as DNA from several fungi, were detected in CSF from ALS patients. Additionally, examination of brain sections from the frontal cortex of ALS patients revealed the existence of immunopositive fungal antigens comprising punctate bodies in the cytoplasm of some neurons. Fungal DNA was also detected in brain tissue using PCR analysis, uncovering the presence of several fungal species. Finally, proteomic analyses of brain tissue demonstrated the occurrence of several fungal peptides. Collectively, our observations provide compelling evidence of fungal infection in the ALS patients analyzed, suggesting that this infection may play a part in the etiology of the disease or may constitute a risk factor for these patients.

Fungal Infection in Patients with Serpiginous Choroiditis or Acute Zonal Occult Outer Retinopathy

ABSTRACT The etiologies of a number of retinopathies, including serpiginous choroiditis and acute zonal occult outer retinopathy (AZOOR), remain uncertain. Recently, we provided evidence that AZOOR is caused by Candida famata infection. The purpose of this article was to investigate the presence of fungal infection in five patients affected with serpiginous choroiditis and five patients with diagnosis of AZOOR. To assess the presence of fungal infection the presence of antibodies in human serum samples against C. famata, C. albicans, C. parapsilosis, C. glabrata and C. krusei was analyzed. In addition, quantitative PCR was carried out to detect fungal genomes in whole blood. Finally, the presence of fungal antigens in the serum samples of patients was investigated. Three AZOOR patients presented high antibody titers against Candida spp., while antibodies against Candida spp. were observed in serum samples from four patients with serpiginous choroiditis. Fungal genomes in peripheral blood were evidenced in serum samples from one AZOOR and four serpiginous choroiditis patients. Fungal antigens were also apparent in the serum of different patients. Our findings indicate that there was evidence of disseminated fungal infection in most patients examined.

Identification of Fungal Species in Brain Tissue from Alzheimer’s Disease by Next-Generation Sequencing

The possibility that patients diagnosed with Alzheimers disease (AD) have disseminated fungal infection has been recently advanced by the demonstration of fungal proteins and DNA in nervous tissue from AD patients. In the present study, next-generation sequencing (NGS) was used to identify fungal species present in the central nervous system (CNS) of AD patients. Initially, DNA was extracted from frozen tissue from four different CNS regions of one AD patient and the fungi in each region were identified by NGS. Notably, whereas a great variety of species were identified using the Illumina platform, Botrytis cinerea and Cryptococcus curvatus were common to all four CNS regions analyzed. Further analysis of entorhinal/cortex hippocampus samples from an additional eight AD patients revealed a variety of fungal species, although some were more prominent than others. Five genera were common to all nine patients: Alternaria, Botrytis, Candida, Cladosporium, and Malassezia. These observations could be used to guide targeted antifungal therapy for AD patients. Moreover, the differences found between the fungal species in each patient may constitute a basis to understand the evolution and severity of clinical symptoms in AD.

Polymicrobial Infections In Brain Tissue From Alzheimer’s Disease Patients

Several studies have advanced the idea that the etiology of Alzheimer’s disease (AD) could be microbial in origin. In the present study, we tested the possibility that polymicrobial infections exist in tissue from the entorhinal cortex/hippocampus region of patients with AD using immunohistochemistry (confocal laser scanning microscopy) and highly sensitive (nested) PCR. We found no evidence for expression of early (ICP0) or late (ICP5) proteins of herpes simplex virus type 1 (HSV-1) in brain sections. A polyclonal antibody against Borrelia detected structures that appeared not related to spirochetes, but rather to fungi. These structures were not found with a monoclonal antibody. Also, Borrelia DNA was undetectable by nested PCR in the ten patients analyzed. By contrast, two independent Chlamydophila antibodies revealed several structures that resembled fungal cells and hyphae, and prokaryotic cells, but most probably were unrelated to Chlamydophila spp. Finally, several structures that could belong to fungi or prokaryotes were detected using peptidoglycan and Clostridium antibodies, and PCR analysis revealed the presence of several bacteria in frozen brain tissue from AD patients. Thus, our results show that polymicrobial infections consisting of fungi and bacteria can be revealed in brain tissue from AD patients.

Corpora Amylacea of Brain Tissue from Neurodegenerative Diseases Are Stained with Specific Antifungal Antibodies.

The origin and potential function of corpora amylacea (CA) remains largely unknown. Low numbers of CA are detected in the aging brain of normal individuals but they are abundant in the central nervous system of patients with neurodegenerative diseases. In the present study, we show that CA from patients diagnosed with Alzheimers disease (AD) contain fungal proteins as detected by immunohistochemistry analyses. Accordingly, CA were labeled with different anti-fungal antibodies at the external surface, whereas the central portion composed of calcium salts contain less proteins. Detection of fungal proteins was achieved using a number of antibodies raised against different fungal species, which indicated cross-reactivity between the fungal proteins present in CA and the antibodies employed. Importantly, these antibodies do not immunoreact with cellular proteins. Additionally, CNS samples from patients diagnosed with amyotrophic lateral sclerosis (ALS) and Parkinsons disease (PD) also contained CA that were immunoreactive with a range of antifungal antibodies. However, CA were less abundant in ALS or PD patients as compared to CNS samples from AD. By contrast, CA from brain tissue of control subjects were almost devoid of fungal immunoreactivity. These observations are consistent with the concept that CA associate with fungal infections and may contribute to the elucidation of the origin of CA.