ALS is characterized by heterogeneous clinical signs and symptoms, and a definite diagnosis may require 13 to 18 months, once of other disorders that can affect upper and lower motor neurons have been excluded 
The identification of specific and sensitive circulating biomarkers for ALS could permit an early diagnosis and avoid unnecessary and potentially harmful therapeutic trials. In particular, an increase in HERVs expression has been observed in some neurological diseases, including ALS, although there is no evidence that this may be the primary causative factor for the pathology.
In a previous study, we documented an HERV-K env antigen humoral immunity response by detecting specific IgG antibodies in serum and CSF of Sardinian patients affected by ALS, with respect to other neurological diseases 
. In this study, to better secure the role of retroviruses in ALS, and validate the use of HERV-K as a possible diagnostic biomarker, we expanded the size of our previously studied cohort to 55, and respective healthy controls to 102, confirming the data obtained in the previous paper 
. We also used different methodological approaches to better understand the role of HERV-K on humoral and cell-mediated immune responses both by using cytometric analysis to quantify the HERV-K env peptides expression on the PBMCs membrane of ALS patients and controls and by investigating HERV-K transcripts in PBMCs by RT-qPCR method. Notably, PBMCs have been proposed as a good non-invasive option for studying ALS 
The analysis of viral protein expression on lymphomonocytes showed that HERV-K env protein was expressed with greater percentage frequency in B cells of ALS patients. This finding supports the results of ELISA tests in serum and CSF showing that the production of specific immunoglobulins towards selective retroviral peptides was significantly higher in patients with ALS with respect to HCs 
. Instead, the analysis of the HERV-K env transcripts has not proven any significant difference between patients and controls as previously observed by Garson 
. Interestingly, other HERVs families have been associated to different neurological diseases, such as HERV-W to multiple sclerosis (MS) from different authors 
, and HERV-W env expression on the membrane of PBMCs in MS patients seems modulated by natalizumab 
. Recently, we have also shown that circulating antibody levels directed against the HERV-W env-su 93–108
and HERV-W env-su 248–262
peptide fragments are different in different CNS demyelinating disorders 
Based on our findings, HERV-K env protein appeared to be poorly expressed on the CD4+ and CD8+ lymphocyte membranes of both ALS patients and controls (data not shown), despite retroviral antigens also determining a humoral response T helps mediate.
The specific immune response which T and B mediated after stimulation of PBMCs with two antigenic peptides derived from the HERV-K env-su glycoprotein, namely HERV-K env 19–37 and HERV-K env 109–126, in patients with ALS and HCs, showed significant differences among the cytokines produced, indicating a good and broad reactivity of the immune system. In particular, the expression of IL-6 by CD19/B cells was significantly different after stimulation with HERV-K env 109–126 peptide in ALS patients in comparison to HCs, suggesting that this peptide is likely responsible for the B-cell activation, considered the autocrine activity of this cytokine. Instead, the HERV-K env 19–37 peptide seems able to foster a pro-inflammatory response by stimulation of CD19/B cells and a statistically significant greater production of IFN-γ. In CD19/B cells, both peptides were not able to stimulate the TNF-α production, while the HERV-K env 19–37 peptide induced a greater production of TNF-α in CD8/T cells.
The mechanism for this latter effect is unknown. We believe it might be due to an insufficient immune innate response in ALS patients after a 6-hour stimulus of PBMCs. This is consistent with an increased retroviral expression observed on monocytes of HCs compared to patients, as TNF-α is a pro-inflammatory cytokine that appears at the early stages of the innate immune response. Previous studies have reported higher levels of IL-6 in ALS patients in comparison to HCs 
and rising IL-6 levels in plasma were also associated with risk for disease progression 
. Interestingly, although IL-6 may have an established bivalent role in inflammation, namely both a pro and anti-inflammatory action, a peripheral IL-6 upregulation usually is related to an inflammatory cell response also able to induce an endothelial cells damage at blood–brain barrier. 
. Thus, our data support the possibility that an increased expression of HERV-K can stimulate IL-6 and IFN-γ determining a chronic inflammatory state through a T lymphocytes/Th1-type cytokines response which, in patients genetically predisposed, may play a role in worsening the disease course.
Moreover, we studied the expression of MIP-1α and MCP-1 by CD19/B-cells stimulated with both peptides, and found a significant increase in the expression of MIP-1α in ALS patients. This finding fits previous data in the literature showing either a negative correlation between expression of MIP-1α and disease progression rate or a positive correlation with disease duration, thus suggesting a possible protective role of this chemokine on ALS outcome 
. The antigenic specific stimulation generated by HERV-K env 19–37
and HERV-K env 109–126
contribute to the specific increase in MIP-1 α levels as observed in serum and CSF by other authors 
. Regarding MCP-1, we document a lower expression by CD19/B cells in ALS patients compared to HCs, following stimulation with both peptides. As higher MCP-1 levels were associated with worse disease severity and faster progression by several authors 
, and due to the HERV-K related lower expression of this chemokine documented in our study, it is likely to work together with the increased expression of MIP-1α to produce a better ALS outcome.
In this context, in order to investigate which IgG subclass of antibodies anti-HERV-K were predominant in patients with ALS, we determined plasma levels of IgG1 and IgG4 immunoglobulins. This is an important issue in order to clarify if the high levels of IgG found in ALS are protective (IgG4) or harmful (IgG1) 
. Indeed, recent data have indicated that IgG4 antibodies may fulfil a protective role dampening the more harmful effects of IgG1 when directed against the same epitopes 
. In addition, we evaluated if the observed IgG1 and IgG4 production could be correlated with disease progression and prognosis. We detected IgG1 plasma levels significantly higher in ALS patients compared to HCs; conversely, no difference between the two groups was observed for IgG4 plasma levels. Of note, IgG1 levels were not significantly different in newly diagnosed ALS patients with respect to long-survivor patients, thus this humoral finding does not have prognostic significance.
Interestingly, in long-survivor ALS patients, IgG1 and IgG4 plasma levels were both significantly increased with respect to HCs. This data indicates that, in long-survivors, the detrimental effect of increased IgG1 levels in plasma may be progressively dampened by the parallel increment in plasma of IgG4 levels. The mechanisms behind the pathological contribution of HERV-K activation in ALS remain yet to be completely clarified. Here, we document divergent mechanisms, related to humoral and cell-mediated immune response to antigenic peptides derived from HERV-K in ALS patients. In this context, our study provides useful information to better understand the possible role of endogenous retroviruses in neurodegenerative diseases. In particular, we highlighted the ability of HERV-K to modulate the immune system at different levels, generating mediators mainly involved in proinflammatory response. Of note, despite depletion, B cells of ALS patients express higher levels of HERV-K env protein compared to HCs feeding the inflammatory loop, by induction of a continuous proinflammatory stimulus of T lymphocytes sustained by exposition of viral protein on B lymphocytes membrane. Furthermore, we better understand the role of the cytokine IL 6 in the early stage of ALS as responsible for the injury of the BBB, such that to support neuronal damage. Certainly, an early knowledge is sorely needed to diagnose ALS, as recently described by Keon et al. 
. Adequate translation of our results into clinical practice may speed up the use of HERV-K as biomarker of disease progression due to its ability to modulate cytokines and chemokines as mediators of inflammation partly responsible for motor neurons damage.