Polymicrobial Sepsis Impairs Antigen-Specific Memory CD4 T Cell-Mediated Immunity.

Patients who survive sepsis display prolonged immune dysfunction and heightened risk of secondary infection. CD4 T cells support a variety of cells required for protective immunity, and perturbations to the CD4 T cell compartment can decrease overall immune system fitness. Using the cecal ligation and puncture (CLP) mouse model of sepsis, we investigated the impact of sepsis on endogenous Ag-specific memory CD4 T cells generated in C57BL/6 (B6) mice infected with attenuated Listeria monocytogenes (Lm) expressing the I-Ab-restricted 2W1S epitope (Lm-2W). The number of 2W1S-specific memory CD4 T cells was significantly reduced on day 2 after sepsis induction, but recovered by day 14. In contrast to the transient numerical change, the 2W1S-specific memory CD4 T cells displayed prolonged functional impairment after sepsis, evidenced by a reduced recall response (proliferation and effector cytokine production) after restimulation with cognate Ag. To define the extent to which the observed functional impairments in the memory CD4 T cells impacts protection to secondary infection, B6 mice were infected with attenuated Salmonella enterica-2W (Se-2W) 30 days before sham or CLP surgery, and then challenged with virulent Se-2W after surgery. Pathogen burden was significantly higher in the CLP-treated mice compared to shams. Similar reductions in functional capacity and protection were noted for the endogenous OVA323-specific memory CD4 T cell population in sepsis survivors upon Lm-OVA challenge. Our data collectively show CLP-induced sepsis alters the number and function of Ag-specific memory CD4 T cells, which contributes (in part) to the characteristic long-lasting immunoparalysis seen after sepsis.

Inhibition of bone morphogenetic protein 6 receptors ameliorates Sjögren's syndrome in mice.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease, with only palliative treatments available. Recent work has suggested that increased bone morphogenetic protein 6 (BMP6) expression could alter cell signaling in the salivary gland (SG) and result in the associated salivary hypofunction. We examined the prevalence of elevated BMP6 expression in a large cohort of pSS patients and tested the therapeutic efficacy of BMP signaling inhibitors in two pSS animal models. Increased BMP6 expression was found in the SGs of 54% of pSS patients, and this increased expression was correlated with low unstimulated whole saliva flow rate. In mouse models of SS, inhibition of BMP6 signaling reduced phosphorylation of SMAD1/5/8 in the mouse submandibular glands, and led to a recovery of SG function and a decrease in inflammatory markers in the mice. The recovery of SG function after inhibition of BMP6 signaling suggests cellular plasticity within the salivary gland and a possibility for therapeutic intervention that can reverse the loss of function in pSS.

Polymicrobial Sepsis Chronic Immunoparalysis Is Defined by Diminished Ag-Specific T Cell-Dependent B Cell Responses.

Immunosuppression is one hallmark of sepsis, decreasing the host response to the primary septic pathogens and/or secondary nosocomial infections. CD4 T cells and B cells are among the array of immune cells that experience reductions in number and function during sepsis. "Help" from follicular helper (Tfh) CD4 T cells to B cells is needed for productive and protective humoral immunity, but there is a paucity of data defining the effect of sepsis on a primary CD4 T cell-dependent B cell response. Using the cecal ligation and puncture (CLP) mouse model of sepsis induction, we observed reduced antibody production in mice challenged with influenza A virus or TNP-KLH in alum early (2 days) and late (30 days) after CLP surgery compared to mice subjected to sham surgery. To better understand how these CD4 T cell-dependent B cell responses were altered by a septic event, we immunized mice with a Complete Freund's Adjuvant emulsion containing the MHC II-restricted peptide 2W1S56-68 coupled to the fluorochrome phycoerythrin (PE). Immunization with 2W1S-PE/CFA results in T cell-dependent B cell activation, giving us the ability to track defined populations of antigen-specific CD4 T cells and B cells responding to the same immunogen in the same mouse. Compared to sham mice, differentiation and class switching in PE-specific B cells were blunted in mice subjected to CLP surgery. Similarly, mice subjected to CLP had reduced expansion of 2W1S-specific T cells and Tfh differentiation after immunization. Our data suggest CLP-induced sepsis impacts humoral immunity by affecting the number and function of both antigen-specific B cells and CD4 Tfh cells, further defining the period of chronic immunoparalysis after sepsis induction.

Enteric immunity, the gut microbiome, and sepsis: Rethinking the germ theory of disease.

Sepsis is a poorly understood syndrome of systemic inflammation responsible for hundreds of thousands of deaths every year. The integrity of the gut epithelium and competence of adaptive immune responses are notoriously compromised during sepsis, and the prevalent assumption in the scientific and medical community is that intestinal commensals have a detrimental role in the systemic inflammation and susceptibility to nosocomial infections seen in critically ill, septic patients. However, breakthroughs in the last decade provide strong credence to the idea that our mucosal microbiome plays an essential role in adaptive immunity, where a human host and its prokaryotic colonists seem to exist in a carefully negotiated armistice with compromises and benefits that go both ways. In this review, we re-examine the notion that intestinal contents are the driving force of critical illness. An overview of the interaction between the microbiome and the immune system is provided, with a special focus on the impact of commensals in priming and the careful balance between normal intestinal flora and pathogenic organisms residing in the gut microbiome. Based on the data in hand, we hypothesize that sepsis induces imbalances in microbial populations residing in the gut, along with compromises in epithelial integrity. As a result, normal antigen sampling becomes impaired, and proliferative cues are intermixed with inhibitory signals. This situates the microbiome, the gut, and its complex immune network of cells and bacteria, at the center of aberrant immune responses during and after sepsis.

Gut Microbial Membership Modulates CD4 T Cell Reconstitution and Function after Sepsis.

Transient lymphopenia is one hallmark of sepsis, and emergent data indicate the CD4 T cell compartment in sepsis survivors is numerically and functionally altered (when examined at the Ag-specific level) compared with nonseptic control subjects. Previous data from our laboratory demonstrated Ag-independent, lymphopenia-induced homeostatic proliferation to be a contributing mechanism by which CD4 T cells numerically recover in sepsis survivors. However, we reasoned it is also formally possible that some CD4 T cells respond directly to Ag expressed by gut-resident microbes released during polymicrobial sepsis. The effect of gut microbiome leakage on CD4 T cells is currently unknown. In this study, we explored the number and function of endogenous CD4 T cells specific for segmented filamentous bacterium (SFB) after cecal ligation and puncture (CLP)-induced sepsis using mice that either contained or lacked SFB as a normal gut-resident microbe. Interestingly, SFB-specific CD4 T cells underwent Ag-driven proliferation in CLP-treated SFB(+), but not in SFB(-), mice. Moreover, CLP-treated SFB(+) mice showed resistance to secondary lethal infection with recombinant SFB Ag-expressing virulent Listeria (but not wild-type virulent Listeria), suggesting the CLP-induced polymicrobial sepsis primed for a protective response by the SFB-specific CD4 T cells. Thus, our data demonstrate that the numerical recovery and functional responsiveness of Ag-specific CD4 T cells in sepsis survivors is, in part, modulated by the intestinal barrier's health discreetly defined by individual bacterial populations of the host's microbiome.

Aquaporin gene therapy corrects Sjögren's syndrome phenotype in mice.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease that is estimated to affect 35 million people worldwide. Currently, no effective treatments exist for Sjögren's syndrome, and there is a limited understanding of the physiological mechanisms associated with xerostomia and hyposalivation. The present work revealed that aquaporin 5 expression, a water channel critical for salivary gland fluid secretion, is regulated by bone morphogenetic protein 6. Increased expression of this cytokine is strongly associated with the most common symptom of primary Sjögren's syndrome, the loss of salivary gland function. This finding led us to develop a therapy in the treatment of Sjögren's syndrome by increasing the water permeability of the gland to restore saliva flow. Our study demonstrates that the targeted increase of gland permeability not only resulted in the restoration of secretory gland function but also resolved the hallmark salivary gland inflammation and systemic inflammation associated with disease. Secretory function also increased in the lacrimal gland, suggesting this local therapy could treat the systemic symptoms associated with primary Sjögren's syndrome.

Alterations in antigen-specific naive CD4 T cell precursors after sepsis impairs their responsiveness to pathogen challenge.

Patients surviving the acute stages of sepsis develop compromised T cell immunity and increased susceptibility to infection. Little is known about the decreased CD4 T cell function after sepsis. We tracked the loss and recovery of endogenous Ag-specific CD4 T cell populations after cecal ligation and puncture-induced sepsis and analyzed the CD4 T cell response to heterologous infection during or after recovery. We observed that the sepsis-induced early loss of CD4 T cells was followed by thymic-independent numerical recovery in the total CD4 T cell compartment. Despite this numerical recovery, we detected alterations in the composition of naive CD4 T cell precursor pools, with sustained quantitative reductions in some populations. Mice that had experienced sepsis and were then challenged with epitope-bearing, heterologous pathogens demonstrated significantly reduced priming of recovery-impaired Ag-specific CD4 T cell responses, with regard to both magnitude of expansion and functional capacity on a per-cell basis, which also correlated with intrinsic changes in Vß clonotype heterogeneity. Our results demonstrate that the recovery of CD4 T cells from sepsis-induced lymphopenia is accompanied by alterations to the composition and function of the Ag-specific CD4 T cell repertoire.

Impact of sepsis on CD4 T cell immunity.

Sepsis remains the primary cause of death from infection in hospital patients, despite improvements in antibiotics and intensive-care practices. Patients who survive severe sepsis can display suppressed immune function, often manifested as an increased susceptibility to (and mortality from) nosocomial infections. Not only is there a significant reduction in the number of various immune cell populations during sepsis, but there is also decreased function in the remaining lymphocytes. Within the immune system, CD4 T cells are important players in the proper development of numerous cellular and humoral immune responses. Despite sufficient clinical evidence of CD4 T cell loss in septic patients of all ages, the impact of sepsis on CD4 T cell responses is not well understood. Recent findings suggest that CD4 T cell impairment is a multipronged problem that results from initial sepsis-induced cell loss. However, the subsequent lymphopenia-induced numerical recovery of the CD4 T cell compartment leads to intrinsic alterations in phenotype and effector function, reduced repertoire diversity, changes in the composition of naive antigen-specific CD4 T cell pools, and changes in the representation of different CD4 T cell subpopulations (e.g., increases in Treg frequency). This review focuses on sepsis-induced alterations within the CD4 T cell compartment that influence the ability of the immune system to control secondary heterologous infections. The understanding of how sepsis affects CD4 T cells through their numerical loss and recovery, as well as function, is important in the development of future treatments designed to restore CD4 T cells to their presepsis state.

Matriptase deletion initiates a Sjögren's syndrome-like disease in mice.

OBJECTIVE: The objective of this study was to determine the effect of epithelial barrier disruption, caused by deficiency of the membrane-anchored serine protease, matriptase, on salivary gland function and the induction of autoimmunity in an animal model. METHODS: Embryonic and acute ablation of matriptase expression in the salivary glands of mice was induced, leading to decreased epithelial barrier function. Mice were characterized for secretory epithelial function and the induction of autoimmunity including salivary and lacrimal gland dysfunction, lymphocytic infiltration, serum anti-Ro/SSA, anti-La/SSB and antinuclear antibodies. Salivary glands immune activation/regulation, barrier function as well as tight junction proteins expression also were determined. Expression of matriptase in minor salivary gland biopsies was compared among pSS patients and healthy volunteers. RESULTS: Embryonic ablation of matriptase expression in mice resulted in the loss of secretory epithelial cell function and the induction of autoimmunity similar to that observed in primary Sjögren's syndrome. Phenotypic changes included exocrine gland dysfunction, lymphocytic infiltrates, production of Sjögren's syndrome-specific autoantibodies, and overall activation of the immune system. Acute ablation of matriptase expression resulted in significant salivary gland dysfunction in the absence of overt immune activation. Analysis of the salivary glands indicates a loss of electrical potential across the epithelial layer as well as altered distribution of a tight junction protein. Moreover, a significant decrease in matriptase gene expression was detected in the minor salivary glands of pSS patients compared with healthy volunteers. CONCLUSIONS: Our findings demonstrate that local impairment of epithelial barrier function can lead to loss of exocrine gland function [corrected] in the absence of inflammation while systemic deletion can induce a primary Sjögren's syndrome like phenotype with autoimmunity and loss of gland function.

Association of bone morphogenetic protein 6 with exocrine gland dysfunction in patients with Sjögren's syndrome and in mice.

OBJECTIVE: Primary Sjögren's syndrome (SS) is characterized by autoimmune activation and loss of function in secretory epithelia. The present study was undertaken to investigate and characterize changes in the epithelia associated with the loss of gland function in primary SS. METHODS: To identify changes in epithelial gene expression, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with primary SS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. The effect of bone morphogenetic protein 6 (BMP-6) on salivary gland function was tested using adeno-associated virus-mediated gene transfer to the salivary glands of C57BL/6 mice. RESULTS: A significant increase in expression of BMP-6 was observed in RNA isolated from SS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector-treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with SS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. CONCLUSION: In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in primary SS, the present results suggest that BMP-6-induced salivary and lacrimal gland dysfunction in primary SS is independent of the autoantibodies and immune activation associated with the disease.

T-cell-mediated immunity and the role of TRAIL in sepsis-induced immunosuppression.

Sepsis is the leading cause of death in most intensive care units, and the death of septic patients usually does not result from the initial septic event but rather from subsequent nosocomial infections. Patients who survive severe sepsis often display severely compromised immune function. Not only is there significant apoptosis of lymphoid and myeloid cells that depletes critical components of the immune system during sepsis, there is also decreased function of the remaining immune cells. Studies of animals and humans suggest the immune defects that occur during sepsis may be critical to pathogenesis and subsequent mortality. This review focuses on sepsis-induced alterations with the cluster differentiation (CD) 8 T-cell compartment that can affect the control of secondary heterologous infections. Understanding how a septic event directly influences CD8 T-cell populations through apoptotic death and homeostatic proliferation and indirectly by immune-mediated suppression will provide valuable starting points for developing new treatment options.

STAT-3 and ERK 1/2 phosphorylation are critical for T-cell alloactivation and graft-versus-host disease.

Graft-versus-host disease (GVHD) is a serious complication of allogeneic bone marrow transplantation, and donor T cells are indispensable for GVHD. Current therapies have limited efficacy, selectivity, and high toxicities. We used a novel flow cytometry technique for the analysis of intracellular phosphorylation events in single cells in murine BMT models to identify and validate novel GVHD drug targets.(1-7) This method circumvents the requirement for large numbers of purified cells, unlike western blots. We defined a signaling profile for alloactivated T cells in vivo and identified the phosphorylation of ERK1/2 and STAT-3 as important events during T-cell (allo)activation in GVHD. We establish that interference with STAT-3 phosphorylation can inhibit T-cell activation and proliferation in vitro and GVHD in vivo. This suggests that phospho-specific flow cytometry is useful for the identification of promising drug targets, and ERK1/2 and STAT-3 phosphorylation in alloactivated T cells may be important for GVHD.

Tumor immunotherapy across MHC barriers using allogeneic T-cell precursors.

We present a strategy for adoptive immunotherapy using T-lineage committed lymphoid precursor cells generated by Notch1-based culture. We found that allogeneic T-cell precursors can be transferred to irradiated individuals irrespective of major histocompatibility complex (MHC) disparities and give rise to host-MHC restricted and host-tolerant functional allogeneic T cells, improving survival in irradiated recipients as well as enhancing anti-tumor responses. T-cell precursors transduced to express a chimeric receptor targeting hCD19 resulted in significant additional anti-tumor activity, demonstrating the feasibility of genetic engineering of these cells. We conclude that ex vivo generated MHC-disparate T-cell precursors from any donor can be used universally for 'off-the-shelf' immunotherapy, and can be further enhanced by genetic engineering for targeted immunotherapy.

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation.

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.

[Impotence and cardiovascular disease: a new perspective in the health-care compromise of the urologist]. / Impotencia y enfermedad cardiovascular: una nueva perspectiva en el compromiso asistencial del urólogo.

OBJECTIVES: To review the evidence currently available to establish the pathogenic connection between erectile dysfunction (ED) and cardiovascular disease (CVD), and the effects this scientific progress has introduced in the classical impotence urology clinic. METHODS: We reviewed the most recent publications about this disease (2000-2004) and the concept of endothelial dysfunction by appropriate MEDLINE searches, with specific selection of reviews and clinical practice guidelines. RESULTS: The fact that ED and CVD share risk factors is confirmed; the pathogenic unity of both processes having endothelial dysfunction as the underlying problem; anticipation of ED over CVD in time of presentation; notable increase of research about this issue over the last two years; the change of scenario in the impotence urology clinics due to these findings. CONCLUSIONS: The number of cases in which ED is not an organ disease but an early symptom of endothelial dysfunction forces changes in the extent and depth of the diagnostic, prognostic and follow-up strategies in the urology impotence clinics of extraordinary importance from both the individual and health-care politics point of view.

[Upper urinary tract video-urodynamics. Current indications]. / "Vidaus" videourodinamia del aparato urinario superior. Indicaciones actuales.

OBJECTIVES: Upper urinary tract videourodynamic studies were first described in the second half of the 20th century (years 60-70). The antegrade pyelogram with constant pressure and controlled flow received the name of its author, "Vela Navarrete test" (1982), who currently refers to it as video-urodynamic study of the upper urinary tract. It is the simultaneous study of pressure, flow, and dynamic x-ray of the upper urinary tract and it keeps same indications than the original design, in spite of the appearance of new dynamic and static imaging techniques (ultrasound, CT scan, MRI, radioisotope studies). The continued practice of video urodynamic study of the upper urinary tract in the FJD prompted an update of the procedure reviewing its current indications. METHODS: Patient is positioned in the prone decubitus on an x-ray table with video. After punctioning renal cavities with fine needle, video urodynamic tests are performed: pyelomanometry, basal pyelic pressure (basic urodynamics study), pressure changes after induced diuresis, and pressure-flow studies. 10-20 ml of urine are obtained for biochemical tests (creatinine, electrolytes, osmolarity,...), which give information about the functional viability of the renal unit, cytology or microbiological tests. Finally, the study concludes with an antegrade pyelogram, a fluoroscopic study of the radiological anatomy which provides morphologic and dynamic data of the upper urinary tract. In the Urodynamics Unit of the FJD, urologists perform 30-40 studies per year. We analyzed retrospectively all studies performed over the last five years (1999-2005) for various pathological entities of kidney dilation. (Table I). RESULTS: Structural and dynamic data obtained by the studies offered conclusive information in most of them facilitating the decision for surgery or observation (Table II). CONCLUSIONS: The interpretation of the upper urinary tract video-urodynamic study is based on radiological and dynamic findings. Flow-controlled pyelogram enables determining the existence of obstruction, dilation volume, and ureteral peristaltic behaviour. Constant pressure enables quantification of obstruction. The video urodynamic study of the upper urinary tract is easy to perform, well tolerated and may be repeated as many times as required, mainly if nephrostomy tube is placed for more prolonged evaluations. It offers excellent anatomical, dynamic, and etiological information about the upper urinary tract, and furthermore conclusive, with minimal risk.

[Current indications of percutaneous nephrostomy associated with extracorporeal shock wave lithotripsy in the treatment of renoureteral stones]. / Indicaciones actuales de la nefrostomía percutanea asociada a la litotricia extracorpórea por ondas de choque en el tratamiento de la litiasis reno ureteral.

OBJECTIVES: To evaluate the usefulness of PN as urinary diversion in the treatment of urinary stones by ESWL. METHODS: Between April 1996 and June 2003 9554 ESWL were performed at the lithiasis unit of the Fundación Jiménez Diaz; 0.91% required previous insertion of a PN. We performed a retrospective analysis of the 49 patients with the diagnosis of upper urinary tract stones who require treatment by ESWL associated with PN. Indications for PN were: ureteral obstruction by fragments 6%, treatment of residual stones after percutaneous nephrolithectomy 6%, ureteral obstruction by a calcified double J stent 5%, urinary sepsis 75%, and obstructive anuria in a solitary kidney 8%. RESULTS: Overall, 87 sessions were performed in 49 patients. Number of shock waves 3996, Kv 7.69. Results where comparable in terms of stone size and composition. 57% of the patients were stone-free after one session, 24% after 2, and 19% required more than 2 sessions. There were two failures requiring surgery. CONCLUSIONS: Although in-situ ESWL is the treatment of choice for renoureteral lithiasis, the PN is a complementary procedure when ureteral obstruction requires treatment; it is non invasive and may be successfully associated to ESWL.