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

Alonso R, Pisa D, Marina AI, Morato E, Rábano A, Rodal I, Carrasco L - Int. J. Biol. Sci. (2015)

Bottom Line: ALS is the most common form of motor neuron disease; yet, to date, the exact etiology of ALS remains unknown.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.

View Article: PubMed Central - PubMed

Affiliation: 1. Centro de Biología Molecular "Severo Ochoa". c/Nicolás Cabrera, 1. Universidad Autónoma de Madrid. Cantoblanco. 28049 Madrid. Spain.

ABSTRACT
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.

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Analysis of C. albicans and enolase antigens in CSF by slot-blot. 20 μl CSF samples were diluted with 180 μl TBS and were blotted onto a nitrocellulose membrane, which was incubated with the rabbit antiserum against C. albicans or enolase or recombinant MBP-enolase (primary antibody) as indicated and afterwards incubated with a rabbit anti rat IgG (secondary antibody). Positive control: control + 200 ng yeast protein or purified MBP-enolase. Negative control: control - corresponds to TBS alone.
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Figure 1: Analysis of C. albicans and enolase antigens in CSF by slot-blot. 20 μl CSF samples were diluted with 180 μl TBS and were blotted onto a nitrocellulose membrane, which was incubated with the rabbit antiserum against C. albicans or enolase or recombinant MBP-enolase (primary antibody) as indicated and afterwards incubated with a rabbit anti rat IgG (secondary antibody). Positive control: control + 200 ng yeast protein or purified MBP-enolase. Negative control: control - corresponds to TBS alone.

Mentions: A highly sensitive slot-blot assay to analyze the presence of fungi in blood serum or CSF has been developed using specific antibodies against fungal antigens 20, 28, 31. Samples are blotted onto a nitrocellulose membrane and then incubated with the corresponding primary antibody at the indicated dilution. Subsequently, the membrane is incubated with a peroxidase-conjugated secondary antibody and the film obtained is scanned. We have previously established the cut-off values to determine negative or positive results 31. We first tested a number of antibodies raised against total fungal cells or purified enolase from C. famata, or a β-tubulin peptide specific for fungi. CSF samples from five ALS patients and from three controls (see Supplementary Table SI) were tested. Figure 1 shows representative results obtained with anti-C. albicans and enolase antibodies. A positive control using yeast proteins and a negative control containing TBS were used. Certainly, CSF samples from ALS patients rendered a clear positive result. Densitometric quantification of this assay employed a panel of different antibodies is presented in Table I indicating the positive reactivity for fungal antigens in all five patients. As expected, values obtained in control CSF samples for a range of fungi were rather low. In contrast, slot-blot values from CSF of ALS patients were generally considerably higher. For example, CSF from patient 1 exhibited high values with several antibodies employed, including anti-C. famata, C. albicans, C. glabrata, enolase and β-tubulin. Further, high values were obtained in most cases with the anti-C. albicans, enolase and β-tubulin antibodies. Not all the antibodies exhibited a similar behavior, since in some instances the differences found with a given antibody were more pronounced between ALS and control CSFs. This may indicate that the fungal antigens present in ALS CSF immunoreact much better than other anti-fungal antibodies. For example, this is the case of values obtained with anti-C. albicans antibodies that rendered a median of 131.2 in ALS and 11.3 in controls, with a P=0.18 and the odds ratio of 8.0. Considering all the values of Table I, the results obtained are: P=0.01 and odds ratio=4.8. Overall, these results indicate that fungal antigens are present in CSF from ALS patients.


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

Alonso R, Pisa D, Marina AI, Morato E, Rábano A, Rodal I, Carrasco L - Int. J. Biol. Sci. (2015)

Analysis of C. albicans and enolase antigens in CSF by slot-blot. 20 μl CSF samples were diluted with 180 μl TBS and were blotted onto a nitrocellulose membrane, which was incubated with the rabbit antiserum against C. albicans or enolase or recombinant MBP-enolase (primary antibody) as indicated and afterwards incubated with a rabbit anti rat IgG (secondary antibody). Positive control: control + 200 ng yeast protein or purified MBP-enolase. Negative control: control - corresponds to TBS alone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4400386&req=5

Figure 1: Analysis of C. albicans and enolase antigens in CSF by slot-blot. 20 μl CSF samples were diluted with 180 μl TBS and were blotted onto a nitrocellulose membrane, which was incubated with the rabbit antiserum against C. albicans or enolase or recombinant MBP-enolase (primary antibody) as indicated and afterwards incubated with a rabbit anti rat IgG (secondary antibody). Positive control: control + 200 ng yeast protein or purified MBP-enolase. Negative control: control - corresponds to TBS alone.
Mentions: A highly sensitive slot-blot assay to analyze the presence of fungi in blood serum or CSF has been developed using specific antibodies against fungal antigens 20, 28, 31. Samples are blotted onto a nitrocellulose membrane and then incubated with the corresponding primary antibody at the indicated dilution. Subsequently, the membrane is incubated with a peroxidase-conjugated secondary antibody and the film obtained is scanned. We have previously established the cut-off values to determine negative or positive results 31. We first tested a number of antibodies raised against total fungal cells or purified enolase from C. famata, or a β-tubulin peptide specific for fungi. CSF samples from five ALS patients and from three controls (see Supplementary Table SI) were tested. Figure 1 shows representative results obtained with anti-C. albicans and enolase antibodies. A positive control using yeast proteins and a negative control containing TBS were used. Certainly, CSF samples from ALS patients rendered a clear positive result. Densitometric quantification of this assay employed a panel of different antibodies is presented in Table I indicating the positive reactivity for fungal antigens in all five patients. As expected, values obtained in control CSF samples for a range of fungi were rather low. In contrast, slot-blot values from CSF of ALS patients were generally considerably higher. For example, CSF from patient 1 exhibited high values with several antibodies employed, including anti-C. famata, C. albicans, C. glabrata, enolase and β-tubulin. Further, high values were obtained in most cases with the anti-C. albicans, enolase and β-tubulin antibodies. Not all the antibodies exhibited a similar behavior, since in some instances the differences found with a given antibody were more pronounced between ALS and control CSFs. This may indicate that the fungal antigens present in ALS CSF immunoreact much better than other anti-fungal antibodies. For example, this is the case of values obtained with anti-C. albicans antibodies that rendered a median of 131.2 in ALS and 11.3 in controls, with a P=0.18 and the odds ratio of 8.0. Considering all the values of Table I, the results obtained are: P=0.01 and odds ratio=4.8. Overall, these results indicate that fungal antigens are present in CSF from ALS patients.

Bottom Line: ALS is the most common form of motor neuron disease; yet, to date, the exact etiology of ALS remains unknown.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.

View Article: PubMed Central - PubMed

Affiliation: 1. Centro de Biología Molecular "Severo Ochoa". c/Nicolás Cabrera, 1. Universidad Autónoma de Madrid. Cantoblanco. 28049 Madrid. Spain.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus