Antonio Clemente

Intraspecific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp. nov. (Pythiaceae)

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1 alpha and beta-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.

Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.

Peptide–MHC-based nanomedicines for autoimmunity function as T-cell receptor microclustering devices

We have shown that nanoparticles (NPs) can be used as ligand-multimerization platforms to activate specific cellular receptors in vivo. Nanoparticles coated with autoimmune disease-relevant peptide-major histocompatibility complexes (pMHC) blunted autoimmune responses by triggering the differentiation and expansion of antigen-specific regulatory T cells in vivo. Here, we define the engineering principles impacting biological activity, detail a synthesis process yielding safe and stable compounds, and visualize how these nanomedicines interact with cognate T cells. We find that the triggering properties of pMHC-NPs are a function of pMHC intermolecular distance and involve the sustained assembly of large antigen receptor microclusters on murine and human cognate T cells. These compounds show no off-target toxicity in zebrafish embryos, do not cause haematological, biochemical or histological abnormalities, and are rapidly captured by phagocytes or processed by the hepatobiliary system. This work lays the groundwork for the design of ligand-based NP formulations to re-program in vivo cellular responses using nanotechnology.

B cells from common variable immunodeficiency patients fail to differentiate to antibody secreting cells in response to TLR9 ligand (CpG-ODN) or anti-CD40+IL21.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterised by hypogammaglobulinaemia and antibody deficiency to T dependent and independent antigens. Patients suffer from recurrent respiratory infections and poor response to vaccination. Although the underlying molecular defect is unknown, most CVID patients show impaired late B cell differentiation. We investigated B cell differentiation and immunoglobulin secretion induced by two different stimuli: TLR9 specific ligand (CpG-ODN) and anti-CD40 combined with IL21. The contribution of BCR signalling (anti-IgM stimulation) was also evaluated. B cells from CVID patients produced low levels of IgG and IgA in response to both kinds of stimuli that was not restored by anti-IgM. Production of IgM was conserved when cells were stimulated with anti-CD40 and IL21. These results point to a wide signalling defect in B lymphocytes from CVID patients that may be related to their hypogammaglobulinaemia and poor response to vaccination.

Phenotype markers and cytokine intracellular production by CD8+ γδ T lymphocytes do not support a regulatory T profile in Behçet's disease patients and healthy controls

gammadelta T lymphocytes (GD) have been suggested as one of the causes of cytokine dysregulation that results in neutrophils hyperactivation in Behçets disease (BD) patients. In addition, GD can provoke cytotoxic lesions in autoimmune diseases by interaction with MICA (MHC class I chain-related A) molecules, through NKG2D receptor on its surface. In contrast, the CD8+ subset of gammadelta T lymphocytes (GDCD8+) has been related to regulatory T activity. The aim of this study was to determine the phenotype and the intracellular cytokine profile in GD from peripheral blood, to discern if they were skewed to an effector or regulatory pattern in BD. We performed phenotype analysis, by three-colour flow cytometry, in 28 BD, 15 healthy controls (HC) and 14 patients with recurrent bucal ulcers (RBU). We studied intracellular cytokine production in 10 BD and 14 HC, after polyclonal stimulation. In addition, we analysed serum IL-15 and soluble MICA, by ELISA, in 27 BD, 21 HC and 40 rheumatoid arthritis patients. The hallmark in BD was a specific increase in CD8 expression by GD, and in GDCD8+ absolute numbers. Most of GDCD8+ presented CD8 alphaalpha homodimers and were negative for CD103, Foxp3 and CTLA-4. GDCD8+ and GDCD8- were high IFNgamma-, but poor IL-2, IL-10, TGFbeta and IL-4-producing cells, with no differences between BD and HC. NKG2D expression on CD8+ T cells, serum IL-15 and soluble MICA were not significantly increased in BD. Our results do not suggest a T regulatory profile for GDCD8+ neither in HC, nor in BD. We cannot rule out other suppression mechanisms or some heterogeneity within this subset that could contribute to regulatory function.

Expansion of myeloid-derived suppressor cells in chronic obstructive pulmonary disease and lung cancer: potential link between inflammation and cancer

AbstractBackground Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer (LC). Myeloid-derived suppressor cells (MDSCs) down-regulate the T cell receptor ζ chain (TCR ζ) through l-arginine deprivation and lead to T cell dysfunction and deficient antitumor immunity. We hypothesized that abnormally high levels of MDSCs in COPD patients may alter tumor immunosurveillance.MethodsWe compared the proportion of circulating MDSCs (Lin-HLA-DR-/CD33+/CD11b+) (by flow cytometry), arginase I (ARG I) serum levels (by ELISA), and expression levels of TCR ζ on circulating lymphocytes (by flow cytometry) in 28 patients with LC, 62 subjects with COPD, 41 patients with both LC and COPD, 40 smokers with normal spirometry and 33 non-smoking controls. T cell proliferation assays were performed in a subgroup of participants (CFSE dilution protocol).ResultsWe found that: (1) circulating MDSCs were up-regulated in COPD and LC patients (with and without COPD); (2) MDSCs expansion was associated with TCR ζ down-regulation in the three groups; (3) in LC patients, these findings were independent of COPD and tobacco smoking exposure; (4) TCR ζ down-regulation correlates with T cell hyporesponsiveness in COPD and LC patients.ConclusionsThese results suggest that tumor immunosurveillance might be impaired in COPD and may contribute to the increased risk of LC reported in these patients.

Dual Cases of Type 1 Narcolepsy with Schizophrenia and Other Psychotic Disorders

OBJECTIVE Cases of narcolepsy in association with psychotic features have been reported but never fully characterized. These patients present diagnostic and treatment challenges and may shed new light on immune associations in schizophrenia. METHOD Our case series was gathered at two narcolepsy specialty centers over a 9-year period. A questionnaire was created to improve diagnosis of schizophrenia or another psychotic disorder in patients with narcolepsy. Pathophysiological investigations included full HLA Class I and II typing, testing for known systemic and intracellular/synaptic neuronal antibodies, recently described neuronal surface antibodies, and immunocytochemistry on brain sections to detect new antigens. RESULTS Ten cases were identified, one with schizoaffective disorder, one with delusional disorder, two with schizophreniform disorder, and 6 with schizophrenia. In all cases, narcolepsy manifested first in childhood or adolescence, followed by psychotic symptoms after a variable interval. These patients had auditory hallucinations, which was the most differentiating clinical feature in comparison to narcolepsy patients without psychosis. Narcolepsy therapy may have played a role in triggering psychotic symptoms but these did not reverse with changes in narcolepsy medications. Response to antipsychotic treatment was variable. Pathophysiological studies did not reveal any known autoantibodies or unusual brain immunostaining pattern. No strong HLA association outside of HLA DQB1*06:02 was found, although increased DRB3*03 and DPA1*02:01 was notable. CONCLUSION Narcolepsy can occur in association with schizophrenia, with significant diagnostic and therapeutic challenges. Dual cases maybe under diagnosed, as onset is unusually early, often in childhood. Narcolepsy and psychosis may share an autoimmune pathology; thus, further investigations in larger samples are warranted.

CD27+ B cells from a subgroup of common variable immunodeficiency patients are less sensitive to apoptosis rescue regardless of interleukin‐21 signalling

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and recurrent infections. Although the underlying cause is unknown, B cells from most CVID patients fail to differentiate to memory or plasma cells. We investigated if increased apoptosis could influence the fate of B cells. For this purpose we activated purified B lymphocytes of CVID patients with a surrogate T‐dependent (anti‐CD40) or T‐independent [cytosine–phosphate–guanosine oligodeoxynucleotides (CpG‐ODN) or anti‐immunoglobulin (Ig)M)] stimulus with or without interleukin (IL)‐21. We found that CD27+ B cells were more sensitive than CD27– B cells to spontaneous apoptosis and less sensitive to rescue from apoptosis. The addition of IL‐21 down‐modulated the protective effect of all the stimuli on CD27– B cells and the protective effect of CpG‐ODN and anti‐IgM on CD27+ B cells. In contrast, IL‐21 rescued unstimulated CD27– B cells and improved the rescue of anti‐CD40‐stimulated CD27+ B cells. When we compared patients and controls, mainly CD27+ B cells from MB0 patients were less sensitive to rescue from apoptosis than those from MB1 patients and controls after activation, irrespective of the IL‐21 effect. Increased apoptosis during an immune response could result in lower levels of immunoglobulin production in these patients.

Follicular T Cells from smB− Common Variable Immunodeficiency Patients Are Skewed Toward a Th1 Phenotype

Germinal center follicular T helper (GCTfh) cells are essential players in the differentiation of B cells. Circulating follicular T helper (cTfh) cells share phenotypic and functional properties with GCTfh cells. Distinct subpopulations of cTfh with different helper capabilities toward B cells can be identified: cTfh1 (CXCR3+CCR6−), cTfh2 (CXCR3−CCR6−), and cTfh17 (CXCR3−CCR6+). Alterations in cTfh function and/or distribution have been associated with autoimmunity, infectious diseases, and more recently, with several monogenic immunodeficiencies. Common variable immunodeficiency (CVID) disease is the commonest symptomatic primary immunodeficiency with a genetic cause identified in only 2–10% of patients. Although a heterogeneous disease, most patients show a characteristic defective B cell differentiation into memory B cells or antibody-secreting cells. We investigated if alterations in CVID cTfh cells frequency or distribution into cTfh1, cTfh2, and cTfh17 subpopulations and regulatory follicular T (Tfr) cells could be related to defects in CVID B cells. We found increased percentages of cTfh exhibiting higher programmed death-1 expression and altered subpopulations distribution in smB− CVID patients. In contrast to smB+ patients and controls, cTfh from smB− CVID patients show increased cTfh1 and decreased cTfh17 subpopulation percentages and increased CXCR3+CCR6+ cTfh, a population analogous to the recently described pathogenic Th17.1. Moreover, Tfr cells are remarkably decreased only in smB− CVID patients. In conclusion, increased cTfh17.1 and cTfh1/cTfh17 ratio in CVID patients could influence B cell fate in smB− CVID patients, with a more compromised B cell compartment, and the decrease in Tfr cells may lead to high risk of autoimmune conditions in CVID patients.

Anti-NMDAR antibodies in new-onset psychosis. Positive results in an HIV-infected patient

The role of neuronal surface autoantibodies (NSAs) in non-encephalitic psychosis is of recent and controversial interest. Most of the studies relating NSAs with psychosis are retrospective and only focused on the N-methyl-d-aspartate glutamate receptor (NMDAR). Our goal was to evaluate the prevalence of IgG antibodies against the NMDAR NR1 subunit (NMDAR-Abs) along with five additional NSAs in 61 first psychotic episode patients and 47 matched controls. We found two patients positive for NMDAR-Abs (3.3%). One of them was eventually considered to have been misdiagnosed and reclassified as encephalitis. The other met the criteria for bipolar I disorder, presented no neurological symptoms and had a comorbid HIV infection of vertical transmission. This is the first reported case of an HIV-infected patient with psychosis associated with NSAs. This study shows that patients presenting with clinically incomplete forms of anti-NMDAR encephalitis, with predominant or isolated psychiatric symptoms, can remain undetected if no ancillary tests are performed. To improve patient diagnosis and treatment of individuals with a first psychotic episode, more detailed neurological examinations might be needed. Further studies are required to better clarify the role of NSAs in the neuropsychiatric effects of HIV infection.