Pathophysiological role of respiratory dysbiosis in hospital-acquired pneumonia.

Hospital-acquired pneumonia is a major cause of morbidity and mortality. The incidence of hospital-acquired pneumonia remains high globally and treatment can often be ineffective. Here, we review the available data and unanswered questions surrounding hospital-acquired pneumonia, discuss alterations of the respiratory microbiome and of the mucosal immunity in patients admitted to hospital, and explore potential approaches to stratify patients for tailored treatments. The lungs have been considered a sterile organ for decades because microbiological culture techniques had shown negative results. Culture-independent techniques have shown that healthy lungs harbour a diverse and dynamic ecosystem of bacteria, changing our comprehension of respiratory physiopathology. Understanding dysbiosis of the respiratory microbiome and altered mucosal immunity in patients with critical illness holds great promise to develop targeted host-directed immunotherapy to reduce ineffective treatment, to improve patient outcomes, and to tackle the global threat of resistant bacteria that cause these infections.

IMPACTO EN LA CALIDAD DE VIDA EN PACIENTES CON DERMATITIS ATÓPICA

Resumen Introducción: la dermatitis atópica es una enfermedad inflamatoria crónica de la piel de etiología multifactorial que produce un impacto en la vida del paciente. Resulta de interés conocer cómo valorar la calidad de vida del paciente relacionada con la salud (CVRS), cuando está en tratamiento de mantenimiento para el control de la DA. Objetivo: evaluar la calidad de vida en pacientes con Dermatitis Atópica durante el tratamiento de mantenimiento y el cumplimiento del mismo. Materiales y Métodos: se realizó un estudio epidemiológico, observacional, multicéntrico y transversal. Se analizó a pacientes con diagnóstico clínico de DA, se realizó cuestionarios de calidad de vida (DLQI, CDLQI y IDQOL) y de cumplimiento de tratamiento (Test de Morisky-Green). Resultados: se encuestaron a 41 pacientes. Se observó afección severa el 29%, moderada el 34%, y solo una minoría (5%) no afecto su calidad de vida; casi el 10% presento extremada afectación en calidad de vida. Los niños menores de 5 años tuvieron como predominio gran afectación en la calidad de vida, mientras que en los grupos etarios mayores de 6 años esta afectación resultó moderada. Al evaluar el cumplimiento del tratamiento, el 76% resulto NO cumplidor. De los cumplidores, el 75% presentó gran afectación en la calidad de vida (p:0,05), sin predominio de sexo. Conclusión: en la población estudiada la mayoría presentó una afectación en la calidad de vida moderada a grave. El 76% de la población se consideró como NO cumplidora de tratamiento. De los cumplidores, el 75% presentaba extremada afectación de la calidad de vida.

Summary Introduction: Atopic dermatitis is a chronic inflammatory skin disease of multifactorial etiology that has an impact on the patient's life. It is interesting to know how the patient value their health related quality of life (HRQoL), when they are in maintenance treatment for the control of AD. Objective: to evaluate the quality of life in patients with Atopic Dermatitis during maintenance treatment and its compliance. Materials and Methods: an epidemiological, observational, multicenter and cross-sectional study was carried out. We analyzed patients with a clinical diagnosis of AD, quality of life questionnaires (DLQI, CDLQI and IDQOL) and compliance with treatment (Morisky-Green test). Results: 41 patients were surveyed. A severe condition was observed in 29%, moderate in 34%, and only a minority (5%) did not affect their quality of life; almost 10% presented extreme affectation in quality of life. Children under 5 years of age had a high prevalence of quality of life, whereas in age groups older than 6 years, this affectation was moderate. When evaluating the compliance of the treatment, 76% was NOT compliant. Of the compliers, 75% presented a great impact on the quality of life (p: 0.05), without predominance of sex. Conclusion: in the studied population, the majority presented moderate to severe quality of life. 76% of the population was considered non-compliant of treatment. Of the compliers, 75% presented extreme affectation of the quality of life.

NIK promotes tissue destruction independently of the alternative NF-κB pathway through TNFR1/RIP1-induced apoptosis.

NF-κB-inducing kinase (NIK) is well-known for its role in promoting p100/NF-κB2 processing into p52, a process defined as the alternative, or non-canonical, NF-κB pathway. Here we reveal an unexpected new role of NIK in TNFR1-mediated RIP1-dependent apoptosis, a consequence of TNFR1 activation observed in c-IAP1/2-depleted conditions. We show that NIK stabilization, obtained by activation of the non-death TNFRs Fn14 or LTßR, is required for TNFα-mediated apoptosis. These apoptotic stimuli trigger the depletion of c-IAP1/2, the phosphorylation of RIP1 and the RIP1 kinase-dependent assembly of the RIP1/FADD/caspase-8 complex. In the absence of NIK, the phosphorylation of RIP1 and the formation of RIP1/FADD/caspase-8 complex are compromised while c-IAP1/2 depletion is unaffected. In vitro kinase assays revealed that recombinant RIP1 is a bona fide substrate of NIK. In vivo, we demonstrated the requirement of NIK pro-death function, but not the processing of its substrate p100 into p52, in a mouse model of TNFR1/LTßR-induced thymus involution. In addition, we also highlight a role for NIK in hepatocyte apoptosis in a mouse model of virus-induced TNFR1/RIP1-dependent liver damage. We conclude that NIK not only contributes to lymphoid organogenesis, inflammation and cell survival but also to TNFR1/RIP1-dependent cell death independently of the alternative NF-κB pathway.

New insights into IL-12-mediated tumor suppression.

During the past two decades, interleukin-12 (IL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models. Through pleiotropic effects on different immune cells that form the tumor microenvironment, IL-12 establishes a link between innate and adaptive immunity that involves different immune effector cells and cytokines depending on the type of tumor or the affected tissue. The robust antitumor response exerted by IL-12, however, has not yet been successfully translated into the clinics. The majority of clinical trials involving treatment with IL-12 failed to show sustained antitumor responses and were associated to toxic side effects. Here we discuss the therapeutic effects of IL-12 from preclinical to clinical studies, and will highlight promising strategies to take advantage of the antitumor activity of IL-12 while limiting adverse effects.

PTPN2 controls differentiation of CD4⁺ T cells and limits intestinal inflammation and intestinal dysbiosis.

Loss-of-function variants within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with increased risk for Crohn's disease (CD). A disturbed regulation of T helper (Th) cell responses causing loss of tolerance against self- or commensal-derived antigens and an altered intestinal microbiota plays a pivotal role in CD pathogenesis. Loss of PTPN2 in the T-cell compartment causes enhanced induction of Th1 and Th17 cells, but impaired induction of regulatory T cells (Tregs) in several mouse colitis models, namely acute and chronic dextran sodium sulfate colitis, and T-cell transfer colitis models. This results in increased susceptibility to intestinal inflammation and intestinal dysbiosis which is comparable with that observed in CD patients. We detected inflammatory infiltrates in liver, kidney, and skin and elevated autoantibody levels indicating systemic loss of tolerance in PTPN2-deficient animals. CD patients featuring a loss-of-function PTPN2 variant exhibit enhanced Th1 and Th17 cell, but reduced Treg markers when compared with PTPN2 wild-type patients in serum and intestinal tissue samples. Our data demonstrate that dysfunction of PTPN2 results in aberrant T-cell differentiation and intestinal dysbiosis similar to those observed in human CD. Our findings indicate a novel and crucial role for PTPN2 in chronic intestinal inflammation.

Laser cutting: influence on morphological and physicochemical properties of polyhydroxybutyrate.

Polyhydroxybutyrate (PHB) is a biocompatible and resorbable implant material. For these reasons, it has been used for the fabrication of temporary stents, bone plates, nails and screws (Peng et al. Biomaterials 1996;17:685). In some cases, the brittle mechanical properties of PHB homopolymer limit its application. A typical plasticizer, triethylcitrate (TEC), was used to overcome such limitations by making the material more pliable. In the past few years, CO2-laser cutting of PHB was used in the manufacturing of small medical devices such as stents. Embrittlement of plasticized PHB tubes has been observed, after laser machining. Consequently, the physicochemical and morphological properties of laser-processed surfaces and cut edges of plasticized polymer samples were examined to determine the extent of changes in polymer properties as a result of laser machining. These studies included determination of the depth of the laser-induced heat affected zone by polariscopy of thin polymer sections. Molecular weight changes and changes in the TEC content as a function of distance from the laser-cut edge were determined. In a preliminary test, the cellular response to the processed material was investigated by cell culture study of L929 mouse fibroblasts on laser-machined surfaces. The heat-affected zone was readily classified into four different regions with a total depth of about 60 to 100 microm (Klamp, Master Thesis, University of Rostock, 1998). These results correspond well with the chemical analysis and molecular weight measurements. Furthermore, it was found that cells grew preferentially on the laser-machined area. These findings have significant implications for the manufacture of medical implants from PHB by laser machining.

The clinical course of experimental autoimmune encephalomyelitis and inflammation is controlled by the expression of CD40 within the central nervous system.

Although it is clear that the function of CD40 on peripheral hematopoietic cells is pivotal to the development of autoimmunity, the function of CD40 in autoimmune disease outside this compartment is unresolved. In a model of experimental autoimmune encephalomyelitis (EAE), evidence is presented that CD40-CD154 interactions within the central nervous system (CNS) are critical determinants of disease development and progression. Using bone marrow (BM) chimeric mice, the data suggest that the lack of expression of CD40 by CNS-resident cells diminishes the intensity and duration of myelin oligodendrocyte glycoprotein (MOG)-induced EAE and also reduces the degree of inflammatory cell infiltrates into the CNS. Although CNS inflammation is compromised in the CD40(+/+)-->CD40(-/-) BM chimeric mice, the restricted CD40 expression had no impact on peripheral T cell priming or recall responses. Analysis of RNA expression levels within the CNS demonstrated that encephalitogenic T cells, which entered a CNS environment in which CD40 was absent from parenchymal microglia, could not elicit the expression of chemokines within the CNS. These data provide evidence that CD40 functions outside of the systemic immune compartment to amplify organ-specific autoimmunity.

Caspase 8 expression and signaling in Fas injury-resistant human fetal astrocytes.

Fas (APO-1/CD95) is a cell surface receptor initially identified in lymphoid cells, but more recently detected in the central nervous system under pathological, usually inflammatory, conditions. In most Fas expressing cells, triggering of Fas by its ligand or by antagonistic antibodies leads to apoptosis. Human fetal astrocytes (HFA) constitutively express Fas yet are resistant to cell death following Fas ligation. In the current study, using dissociated cultures of human fetal central nervous system-derived cells, we attempted to identify a basis for HFA resistance to Fas-mediated injury. We compared the components of the Fas signaling pathway of HFA to those of two human cell lines susceptible to Fas-mediated injury, U251 glioma and Jurkat T-cells. We found that HFA did not express caspase 8 (FLICE), the caspase primarily activated on Fas signaling. Although we could induce caspase 8 in HFA with the inflammatory cytokines IFNgamma and TNFalpha, HFA remained resistant to Fas-mediated injury. Addition of inflammatory cytokines to the extracellular milieu also increased FLIP mRNA (FLICE inhibitory protein). Furthermore, upon triggering of cytokine-treated cells with FasL, we observed upregulation of the cleavage product of FLIP (p43-FLIP) previously shown to associate with the DISC and to block caspase 8 recruitment, thereby inhibiting Fas-mediated death. Our findings indicate that caspase 8 and its regulators play a central role in determining the response to Fas ligation of HFA and support a role for Fas signaling in the developing central nervous system other than related to cytotoxicity.

Mechanisms of tissue injury in multiple sclerosis: opportunities for neuroprotective therapy.

Development of neuroprotective therapies for multiple sclerosis is dependent on defining the precise mechanisms whereby immune effector cells and molecules are able to induce relatively selective injury of oligodendrocytes (OLs) and their myelin membranes. The selectivity of this injury could be conferred either by the properties of the effectors or the targets. The former would involve antigen specific recognition by either antibody or T cell receptor of the adaptive immune system. OLs are also susceptible to non antigen restricted injury mediated by components of the innate immune system including macrophages/microglia and NK cells. Target related selectivity could reflect the expression of death inducing surface receptors (such as Fas or TNFR-1) required for interaction with effector mediators and subsequent intracellular signaling pathways, including the caspase cascade. Development of therapeutic delivery systems, which would reach the site of disease activity within the CNS, will permit the administration of inhibitors either of the cell death pathway or of effector target interaction and opens new avenues to neuroprotection approach.

B7 expression and antigen presentation by human brain endothelial cells: requirement for proinflammatory cytokines.

Interaction between systemic immune cells with cells of the blood-brain barrier is a central step in development of CNS-directed immune responses. Endothelial cells are the first cells of the blood-brain barrier encountered by migrating lymphocytes. To investigate the antigen-presenting capacity of human adult brain endothelial cells (HBECs), we used HBECs derived from surgically resected temporal lobe tissue, cocultured with allogeneic peripheral blood derived CD4+ T lymphocytes. HBECs in response to IFN-gamma, but not under basal culture conditions, expressed HLA-DR, B7.1 and B7.2 antigens. Despite such up-regulation, these IFN-gamma-treated HBECs, in contrast to human microglia and PB monocytes, did not sustain allogeneic CD4+ cell proliferation, supported only low levels of IL-2 and IFN-gamma production, and did not stimulate IL-2 receptor expression. CD4+ T cell proliferation and increased IL-2 receptor expression could be obtained by addition of IL-2. Our data suggests that, although HBECs cannot alone support T cell proliferation and cytokine production, HBECs acting in concert with cytokines derived from a proinflammatory environment could support such a response.

Interferon-gamma modulates human oligodendrocyte susceptibility to Fas-mediated apoptosis.

Interferon gamma (IFN-gamma) has been shown to be produced within multiple sclerosis (MS) lesions by infiltrating lymphocytes; systemic administration of this cytokine induces exacerbation of the disease. The aim of the current study was to establish the contribution of IFN-gamma to oligodendrocyte (OL) injury. Our studies utilized cultured human OLs, obtained by dissociation of surgically derived non-MS adult brain tissue. Neither cell survival nor myelin basic protein (MBP) gene expression were affected after 96 hours of treatment with IFN-gamma (100 U/ml), as assessed by LDH release, nucleosome enrichment assay, and RT-PCR. Expression of the death receptor Fas (CD95, APO-1) was, however, significantly increased. Furthermore, IFN-gamma-treated OLs became susceptible to Fas-mediated apoptosis when compared with untreated cells, and were protected by pretreatment with the caspase inhibitor ZVAD. TNF-alpha augmented the IFN-gamma-induced effect. Our results thus indicate that IFN-gamma is not directly cytotoxic for human OLs in culture, but could indirectly modulate functional injury-related responses by upregulating Fas on the cell surface.

Brain-immune connection: immuno-regulatory properties of CNS-resident cells.

Even though the immune privileged status of the central nervous system (CNS) limits access of systemic immune cells through the blood brain barrier (BBB), an immune response can occur in this compartment with or without major breach of the BBB. In this review, we consider properties of resident cells of the CNS, that participate in regulating the neural antigen (Ag)-directed immune responses implicated in autoimmune diseases such as multiple sclerosis (MS). Under such conditions, the CNS is usually viewed as the target or victim of the immune assault, because such immune responses are thought to be initiated and regulated within the systemic immune compartment. The CNS-endogenous cells may themselves, however, initiate, regulate and sustain an immune response. We consider the immune regulatory functions within the CNS in terms of events occurring within the CNS parenchyma (microglia, astroglia) and at the vascular interface. These regulatory functions involve antigen presentation to T cells and polarization of the cytokine response of these cells. Such responses may contribute not only to the overall tissue injury in primary immune disorders but also in a wide range of traumatic, ischemic and degenerative processes.

CD40 engagement stimulates IL-12 p70 production by human microglial cells: basis for Th1 polarization in the CNS.

The APC-derived cytokine interleukin (IL)-12 polarizes CD4 T cells towards the pro-inflammatory Th1 phenotype and has been shown to be crucial for the development of EAE in rodents. In this study we demonstrate that production of IL-12 by human adult CNS-derived microglial cells can be triggered by cell contact with activated T cells. Microglial activation and IL-12 production can be blocked by anti-CD154 mAbs. IL-12 production could also be induced by direct engagement of CD40 on microglia using a CD40 agonist. IL-12 secretion by microglia is significantly reduced by TNF and IFN-gamma antagonists showing that the IL-12 production is subject to regulation by auto- and paracrine stimuli.

Bradykinin B1 receptor expression and function on T lymphocytes in active multiple sclerosis.

BACKGROUND: Lesion development in MS is initiated by migration of inflammatory cells into the central nervous system, a process dependent on endothelial cell-lymphocyte interaction. Bradykinin B1 receptor is a membrane-bound G protein-coupled receptor shown to be upregulated on the surface of various cells types during inflammation. OBJECTIVE: To assess the expression and function of the bradykinin B1 receptor on T lymphocytes from MS patients. METHODS: The authors used multiplex polymerase chain reaction amplification and Western blot techniques to demonstrate B1 receptor expression by T cells. A modified Boyden chamber assay also was used to assess the effect of B1 agonist and antagonist on T cell migration. RESULTS: The authors demonstrated that the expression of B1 receptor was upregulated on T cells derived from peripheral blood of MS patients. Expression of this receptor was upregulated on T cells from patients with secondary progressive MS and relapsing-remitting patients in active relapse. Expression was lower in relapsing remitting patients in remission and least in control subjects, including patients with epilepsy, chronic inflammatory demyelinating polyneuritis, and systemic lupus erythematosus. In vitro treatment of cells from healthy control subjects with tumor necrosis factor-alpha and interferon-gamma also induced the expression of B1 receptors. The authors also found that the significantly higher rate of migration of MS T lymphocytes, compared with control subjects in the Boyden chamber assay, could be prevented by the addition of the selective and stable B1 agonist Sar (D-Phe8) desArg9-BK. CONCLUSION: The authors demonstrate that B1 receptors are upregulated by T lymphocytes during the course of MS and that signaling through this receptor with a B1 agonist can negatively regulate T-cell migration in vitro.

Epipodite and fat cells as sites of hemoglobin synthesis in the branchiopod crustacean Daphnia magna.

In contrast to the malacostracan crustaceans that use hemocyanin as the oxygen carrier, a number of branchiopod crustaceans, such as the water flea Daphnia magna, utilize hemoglobin (Hb) as the respiratory protein. By means of in situ hybridization (ISH) techniques with subsequent signal amplification using catalyzed reporter deposition, sites of Hb synthesis were localized in Daphnia magna. Based on a previously reported Hb-cDNA sequence, a specific ISH probe was designed and hybridized with the Hb-mRNA in histological sections of adult D. magna. The detection of Hb-mRNA was tissue specific and revealed that Hb is synthesized in fat cells, which play a role in fat and glycogen metabolism, and in epithelial cells of the epipodites, which are involved in osmoregulation. Sites of Hb synthesis have been identified in several invertebrate phyla, including Annelida and Nematoda. However, this is the first example in the class Crustacea, and only the second in the phylum Arthropoda.

Expression of a homologue of rat NG2 on human microglia.

The expression of NG2 chondroitin sulfate has been widely associated with oligodendrocyte precursors in rodents. We used a monoclonal antibody (9.2.27) against the human homologue of the rat NG2 to determine whether expression of this molecule was associated with a specific glial cell population present in dissociated cell preparations derived from adult and fetal human brain tissue. Our data, derived using FACS and immunocytochemical analyses of immediately ex vivo or cultured glial cells, indicate that the large majority of NG2 expressing cells belonged to the microglial lineage (CD68, CD11c) rather than to the oligodendrocyte lineage (O4, A2B5, GalC). In situ immunohistochemistry performed on non-fixed normal spinal cord tissue confirmed the observation that NG2 is expressed by mononuclear phagocytes of the CNS. In contrast, peripheral blood-derived monocytes were NG2(-). Cells from fetal brain tissue showed only small numbers of NG2(+) cells, which was consistent with the number of microglial cells in this preparation. In absence of additional markers, we cannot exclude that this anti-NG2 mAb might also recognize human oligodendrocyte progenitor cells.

Interferon-gamma secretion by peripheral blood T-cell subsets in multiple sclerosis: correlation with disease phase and interferon-beta therapy.

Interferon-gamma (IFN-gamma) is implicated as a participant in the immune effector and regulatory mechanisms considered to mediate the pathogenesis of multiple sclerosis (MS). We have used an intracellular cytokine staining technique to demonstrate that the proportion of ex vivo peripheral blood CD4 and CD8 T-cell subsets expressing IFN-gamma is increased in secondary progressing (SP) MS patients, whereas the values in untreated relapsing-remitting (RR) MS patients are reduced compared with those of controls. Patients treated with interferon-beta (IFN-beta) have an even more significant reduction in the percentage of IFN-gamma-secreting cells. The finding that the number of IFN-gamma-expressing CD8 cells is increased in SPMS patients, a group with reduced functional suppressor activity, and is most significantly reduced by IFN-beta therapy, which increases suppressor activity, indicates that IFN-gamma secretion by CD8 T cells and functional suppressor defects attributed to this cell subset in MS can be dissociated.

Inhibition of Th1 polarization by soluble TNF receptor is dependent on antigen-presenting cell-derived IL-12.

Th1-polarized CD4+ T cells are considered central to the development of a number of target-directed autoimmune disorders including multiple sclerosis. The APC-derived cytokine IL-12 is a potent inducer of Th1 polarization in T cells. Inhibition of IL-12 in vivo blocks the development of experimental allergic encephalomyelitis, the animal model for multiple sclerosis. Based on previous work that suggests that the production of IL-12 by activated human central nervous system-derived microglia is regulated by autocrine TNF-alpha, we wanted to determine whether inhibition of TNF could induce a reduction of Th1 responses by its impact on systemic APCs. We found that soluble TNFR p75-IgG fusion protein (TNFR:Fc) inhibited production of IFN-gamma by allo-Ag-activated blood-derived human CD4 T cells. We documented reduced IL-12 p70 production by APCs in the MLR. By adding back recombinant IL-12, we could rescue IFN-gamma production, indicating that TNFR:Fc acts on APC-derived IL-12. Consistent with an inhibition of the Th1 polarization, we found a decreased expression of IL-12R-beta2 subunit on the T cells. Furthermore, the capacity of T cells to secrete IFN-gamma upon restimulation when previously treated with TNFR:Fc is impaired, whereas IL-2 secretion was not altered. Our results define a TNF-dependent cytokine network that favors development of Th1 immune responses.

CD95-CD95L: can the brain learn from the immune system?

Members of the tumor necrosis factor/nerve growth factor receptor superfamily of cell-surface molecules can play the dual role of mediating either cytotoxicity or cell survival, both in the immune system and in the nervous system. A member of this superfamily, CD95 (also known as ApoI or Fas), was initially identified in the immune system and has been shown to mediate receptor-dependent programmed cell death and to be expressed in the nervous system. In neurodegenerative disorders, CD95-CD95 ligand expression on glial cells might precede receptor-mediated apoptosis by cells of the CNS. It is now being recognized that CD95 signaling by immune cells mediates effects other than apoptosis, such as cell survival and under inflammatory conditions expression of this protein promotes neural-immune interactions. Both neuroscientists and immunologists can contribute to defining the mechanisms underlying these divergent effects and utilize such knowledge to aid understanding of cell death and survival.