Impaired Cellular Immunity in Kwashiorkor with Improvement after Therapy

Children with kwashiorkor showed a high incidence of deranged cellular immunity as evidenced by impairment of delayed cutaneous hypersensitivity reactions to candida and diphtheria toxoid antigens and of lymphocyte transformation after phytohaemagglutinin stimulation. This may contribute to their susceptibility to infection. A correlation was shown between the degree of impairment of tests of cellular immunity and the severity of the kwashiorkor. Once recovery was initiated the skin tests gave the expected positive results and the lymphocyte transformation index improved. Protein deprivation may result in impaired deoxyribonucleic acid (DNA) synthesis and in atrophy of both the thymus and the lymphoid tissue.     

Subclinical Thyroid Dysfunction and Cognitive Decline in Old Age

Background

Subclinical thyroid dysfunction has been implicated as a risk factor for cognitive decline in old age, but results are inconsistent. We investigated the association between subclinical thyroid dysfunction and cognitive decline in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER).

Methods

Prospective longitudinal study of men and women aged 70–82 years with pre-existing vascular disease or more than one risk factor to develop this condition (N = 5,154). Participants taking antithyroid medications, thyroid hormone supplementation and/or amiodarone were excluded. Thyroid function was measured at baseline: subclinical hyper- and hypothyroidism were defined as thyroid stimulating hormones (TSH) <0.45 mU/L or >4.50 mU/L respectively, with normal levels of free thyroxine (FT4). Cognitive performance was tested at baseline and at four subsequent time points during a mean follow-up of 3 years, using five neuropsychological performance tests.

Results

Subclinical hyperthyroidism and hypothyroidism were found in 65 and 161 participants, respectively. We found no consistent association of subclinical hyper- or hypothyroidism with altered cognitive performance compared to euthyroid participants on the individual cognitive tests. Similarly, there was no association with rate of cognitive decline during follow-up.

Conclusion

We found no consistent evidence that subclinical hyper- or hypothyroidism contribute to cognitive impairment or decline in old age. Although our data are not in support of treatment of subclinical thyroid dysfunction to prevent cognitive dysfunction in later life, only large randomized controlled trials can provide definitive evidence.     

Immunity,Age and Loss of Immunohomeostasis1

An interpretative discussion on the cellular basis of the association between immunosenescence and loss of immunologic homeostasis was developed from results of studies performed to resolve the issues (a) of whether cells in certain stages of differentiation are more vulnerable to aging than those in other stages and (b) whether the regulatory cells participating in modulating immune responses are prime targets of aging. Young and old mice were exposed to (1) 6-thioguanine (6-TG) to study the activity of their mitotically active and inactive cells and (2) 500R of irradiation to study regeneration of their immune cells. Many alterations were detected in old mice including the existence of a relatively large pool of mitotically inactive pluripotent stem cells in the bone marrow, heightened stem cell regenerative activity, and altered patterns of immunologic regeneration. These kinetic abnormalities reflect age-related changes in stem cells and their progenies, and in the lymphohematopoietic stromal cells.     

Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death

Receptor-interacting protein 1 (RIP1; RIPK1) is a key regulator of multiple signaling pathways that mediate inflammatory responses and cell death. TNF-TNFR1 triggered signaling complex formation, subsequent NF-κB and MAPK activation and induction of cell death involve RIPK1 ubiquitination at several lysine residues including Lys376 and Lys115. Here we show that mutating the ubiquitination site K376 of RIPK1 (K376R) in mice activates cell death resulting in embryonic lethality. In contrast to Ripk1^K376R/K376R mice, Ripk1^K115R/K115R mice reached adulthood and showed slightly higher responsiveness to TNF-induced death. Cell death observed in Ripk1^K376R/K376R embryos relied on RIPK1 kinase activity as administration of RIPK1 inhibitor GNE684 to pregnant heterozygous mice effectively blocked cell death and prolonged survival. Embryonic lethality of Ripk1^K376R/K376R mice was prevented by the loss of TNFR1, or by simultaneous deletion of caspase-8 and RIPK3. Interestingly, elimination of the wild-type allele from adult Ripk1^K376R/cko mice was tolerated. However, adult Ripk1^K376R/cko mice were exquisitely sensitive to TNF-induced hypothermia and associated lethality. Absence of the K376 ubiquitination site diminished K11-linked, K63-linked, and linear ubiquitination of RIPK1, and promoted the assembly of death-inducing cellular complexes, suggesting that multiple ubiquitin linkages contribute to the stability of the RIPK1 signaling complex that stimulates NF-κB and MAPK activation. In contrast, mutating K115 did not affect RIPK1 ubiquitination or TNF stimulated NF-κB and MAPK signaling. Overall, our data indicate that selective impairment of RIPK1 ubiquitination can lower the threshold for RIPK1 activation by TNF resulting in cell death and embryonic lethality.Subject terms: Acute inflammation, Chronic inflammation     

Nitric Oxide Synthase Dysfunction Contributes to Impaired Cerebroarteriolar Reactivity in Experimental Cerebral Malaria

Cerebrovascular dysfunction plays a key role in the pathogenesis of cerebral malaria. In experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA, cerebrovascular dysfunction characterized by vascular constriction, occlusion and damage results in impaired perfusion and reduced cerebral blood flow and oxygenation, and has been linked to low nitric oxide (NO) bioavailability. Here, we directly assessed cerebrovascular function in ECM using a novel cranial window method for intravital microscopy of the pial microcirculation and probed the role of NOS isoforms and phosphorylation patterns in the impaired vascular responses. We show that pial arteriolar responses to endothelial NOS (eNOS) and neuronal NOS (nNOS) agonists (Acetylcholine (ACh) and N-Methyl-D-Aspartate (NMDA)) were blunted in mice with ECM, and could be partially recovered by exogenous supplementation of tetrahydrobiopterin (BH4). Pial arterioles in non-ECM mice infected by Plasmodium berghei NK65 remained relatively responsive to the agonists and were not significantly affected by BH4 treatment. These findings, together with the observed blunting of NO production upon stimulation by the agonists, decrease in total NOS activity, augmentation of lipid peroxidation levels, upregulation of eNOS protein expression, and increase in eNOS and nNOS monomerization in the brain during ECM development strongly indicate a state of eNOS/nNOS uncoupling likely mediated by oxidative stress. Furthermore, the downregulation of Serine 1176 (S1176) phosphorylation of eNOS, which correlated with a decrease in cerebrovascular wall shear stress, implicates hemorheological disturbances in eNOS dysfunction in ECM. Finally, pial arterioles responded to superfusion with the NO donor, S-Nitroso-L-glutathione (GSNO), but with decreased intensity, indicating that not only NO production but also signaling is perturbed during ECM. Therefore, the pathological impairment of eNOS and nNOS functions contribute importantly to cerebrovascular dysfunction in ECM and the recovery of intrinsic functionality of NOS to increase NO bioavailability and restore vascular health represents a target for ECM treatment.     

GPR55 Deletion in Mice Leads to Age-Related Ventricular Dysfunction and Impaired Adrenoceptor-Mediated Inotropic Responses

G protein coupled receptor 55 (GPR55) is expressed throughout the body, and although its exact physiological function is unknown, studies have suggested a role in the cardiovascular system. In particular, GPR55 has been proposed as mediating the haemodynamic effects of a number of atypical cannabinoid ligands; however this data is conflicting. Thus, given the incongruous nature of our understanding of the GPR55 receptor and the relative paucity of literature regarding its role in cardiovascular physiology, this study was carried out to examine the influence of GPR55 on cardiac function. Cardiac function was assessed via pressure volume loop analysis, and cardiac morphology/composition assessed via histological staining, in both wild-type (WT) and GPR55 knockout (GPR55^−/−) mice. Pressure volume loop analysis revealed that basal cardiac function was similar in young WT and GPR55^−/− mice. In contrast, mature GPR55^−/− mice were characterised by both significant ventricular remodelling (reduced left ventricular wall thickness and increased collagen deposition) and systolic dysfunction when compared to age-matched WT mice. In particular, the load-dependent parameter, ejection fraction, and the load-independent indices, end-systolic pressure-volume relationship (ESPVR) and E _max, were all significantly (P<0.05) attenuated in mature GPR55^−/− mice. Furthermore, GPR55^−/− mice at all ages were characterised by a reduced contractile reserve. Our findings demonstrate that mice deficient in GPR55 exhibit maladaptive adrenergic signalling, as evidenced by the reduced contractile reserve. Furthermore, with age these mice are characterised by both significant adverse ventricular remodelling and systolic dysfunction. Taken together, this may suggest a role for GPR55 in the control of adrenergic signalling in the heart and potentially a role for this receptor in the pathogenesis of heart failure.     

Manganese Supplementation Reduces High Glucose-induced Monocyte Adhesion to Endothelial Cells and Endothelial Dysfunction in Zucker Diabetic Fatty Rats

Endothelial dysfunction is a hallmark of increased vascular inflammation, dyslipidemia, and the development of atherosclerosis in diabetes. Previous studies have reported lower levels of Mn²⁺ in the plasma and lymphocytes of diabetic patients and in the heart and aortic tissue of patients with atherosclerosis. This study examines the hypothesis that Mn²⁺ supplementation can reduce the markers/risk factors of endothelial dysfunction in type 2 diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured with or without Mn²⁺ supplementation and then exposed to high glucose (HG, 25 mm) to mimic diabetic conditions. Mn²⁺ supplementation caused a reduction in monocyte adhesion to HUVECs treated with HG or MCP-1. Mn²⁺ also inhibited ROS levels, MCP-1 secretion, and ICAM-1 up-regulation in HUVECs treated with HG. Silencing studies using siRNA against MnSOD showed that similar results were observed in MnSOD knockdown HUVECs following Mn²⁺ supplementation, suggesting that the effect of manganese on monocyte adhesion to endothelial cells is mediated by ROS and ICAM-1, but not MnSOD. To validate the relevance of our findings in vivo, Zucker diabetic fatty rats were gavaged daily with water (placebo) or MnCl₂ (16 mg/kg of body weight) for 7 weeks. When compared with placebo, Mn²⁺-supplemented rats showed lower blood levels of ICAM-1 (17%, p < 0.04), cholesterol (25%, p < 0.05), and MCP-1 (28%, p = 0.25). These in vitro and in vivo studies demonstrate that Mn²⁺ supplementation can down-regulate ICAM-1 expression and ROS independently of MnSOD, leading to a decrease in monocyte adhesion to endothelial cells, and therefore can lower the risk of endothelial dysfunction in diabetes.     

A Common Mutation in Paraoxonase-2 Results in Impaired Lactonase Activity

Paraoxonases (PONs) are a family of lactonases with promiscuous enzyme activity that has been implicated in multiple diseases. PON2 is intracellularly located, is the most ubiquitously expressed PON, and has the highest lactonase activity of the PON family members. Whereas some single-nucleotide polymorphisms (SNPs) in PON1 have resulted in altered enzymatic activity in serum, to date the functional consequences of SNPs on PON2 function remain unknown. We hypothesized that a common PON2 SNP would result in impaired lactonase activity. Substitution of cysteine for serine at codon 311 in recombinant PON2 resulted in normal protein production and localization but altered glycosylation and decreased lactonase activity. Moreover, we screened 200 human lung samples for the PON2 Cys³¹¹ variant and found that in vivo this mutation impaired lactonase activity. These data suggest that impaired lactonase activity may play a role in innate immunity, atherosclerosis, and other diseases associated with the PON2 311 SNP.     

Impaired Epstein-Barr Virus-Specific Neutralizing Antibody Response during Acute Infectious Mononucleosis Is Coincident with Global B-Cell Dysfunction

Here we present evidence for previously unappreciated B-cell immune dysregulation during acute Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). Longitudinal analyses revealed that patients with acute IM have undetectable EBV-specific neutralizing antibodies and gp350-specific B-cell responses, which were associated with a significant reduction in memory B cells and no evidence of circulating antibody-secreting cells. These observations correlate with dysregulation of tumor necrosis factor family members BAFF and APRIL and increased expression of FAS on circulating B cells.     

The Absence of CCR7 Results in Dysregulated Monocyte Migration and Immunosuppression Facilitating Chronic Cutaneous Leishmaniasis

The protozoan parasite Leishmania major causes cutaneous lesions to develop at the site of infection, which are resolved with a strong Th1 immune response in resistant hosts, such as C57BL/6 mice. In contrast, the lesions ulcerate in susceptible hosts which display a Th2 response, such as BALB/c mice. The migration of cells in the immune response to L. major is regulated by chemokines and their receptors. The chemokine receptor CCR7 is expressed on activated DCs and naïve T cells, allowing them to migrate to the correct micro-anatomical positions within secondary lymphoid organs. While there have been many studies on the function of CCR7 during homeostasis or using model antigens, there are very few studies on the role of CCR7 during infection. In this study, we show that B6.CCR7^-/- mice were unable to resolve the lesion and developed a chronic disease. The composition of the local infiltrate at the lesion was significantly skewed toward neutrophils while the proportion of CCR2⁺ monocytes was reduced. Furthermore, a greater percentage of CCR2⁺ monocytes expressed CCR7 in the footpad than in the lymph node or spleen of B6.WT mice. We also found an increased percentage of regulatory T cells in the draining lymph node of B6.CCR7^-/- mice throughout infection. Additionally, the cytokine milieu of the lymph node showed a Th2 bias, rather than the resistant Th1 phenotype. This data shows that CCR7 is required for a protective immune response to intracellular L. major infection.     

Murine Hyaluronidase 2 Deficiency Results in Extracellular Hyaluronan Accumulation and Severe Cardiopulmonary Dysfunction

Hyaluronidase (HYAL) 2 is a membrane-anchored protein that is proposed to hydrolyze hyaluronan (HA) to smaller fragments that are internalized for breakdown. Initial studies of a Hyal2 knock-out (KO) mouse revealed a mild phenotype with high serum HA, supporting a role for HYAL2 in HA breakdown. We now describe a severe cardiac phenotype, deemed acute, in 54% of Hyal2 KO mice on an outbred background; Hyal2 KO mice without the severe cardiac phenotype were designated non-acute. Histological studies of the heart revealed that the valves of all Hyal2 KO mice were expanded and the extracellular matrix was disorganized. HA was detected throughout the expanded valves, and electron microscopy confirmed that the accumulating material, presumed to be HA, was extracellular. Both acute and non-acute Hyal2 KO mice also exhibited increased HA in the interstitial extracellular matrix of atrial cardiomyocytes compared with control mice. Consistent with the changes in heart structure, upper ventricular cardiomyocytes in acute Hyal2 KO mice demonstrated significant hypertrophy compared with non-acute KO and control mice. When the lungs were examined, evidence of severe fibrosis was detected in acute Hyal2 KO mice but not in non-acute Hyal2 KO or control mice. Total serum and heart HA levels, as well as size, were increased in acute and non-acute Hyal2 KO mice compared with control mice. These findings indicate that HYAL2 is essential for the breakdown of extracellular HA. In its absence, extracellular HA accumulates and, in some cases, can lead to cardiopulmonary dysfunction. Alterations in HYAL2 function should be considered as a potential contributor to cardiac pathologies in humans.     

Advanced Parental Age Impaired Fear Conditioning and Hippocampal LTD in Adult Female Rat Offspring

Advanced maternal or paternal age is associated with increased risks of cognitive and emotional disorders. Chronic stress is also a common experience in human life that causes psychiatric diseases. However, the synergistic effects of these two factors on offspring are rarely studied. In the present study, the offspring of both young (3–4 months) and old (12–14 months) rat parents were given CUMS for 21 days at the age of 4 weeks. The effects of advanced parental age and chronic unpredictable mild stress (CUMS) on emotional and cognitive behaviors and the related cellular mechanisms were investigated by using behavioral and electrophysiological techniques. We found that CUMS decreased sucrose consumption, increased anxiety, and impaired learning and memory in offspring from both old and young breeders. However, advanced parental age impaired fear memory and spatial memory mainly in female offspring. The serum corticosterone of female offspring was lower than males, but advanced parental age significantly elevated serum corticosterone in female offspring in response to electrical foot shocks. In addition, hippocampal LTD was severely impaired in female offspring from older parents. Our results indicated that female offspring from older breeders might be more sensitive to stress, and the hippocampal function was more vulnerable. These results might provide experimental basis for the prevention and treatment of advanced parental age related psychiatric disorders in future.     

Chromosome Instability Underlies Hematopoietic Stem Cell Dysfunction and Lymphoid Neoplasia Associated with Impaired Fbw7-Mediated Cyclin E Regulation

The Fbw7 ubiquitin ligase critically regulates hematopoietic stem cell (HSC) function, though the precise contribution of individual substrate ubiquitination pathways to HSC homeostasis is unknown. In the work reported here, we used a mouse model in which we introduced two knock-in mutations (T74A and T393A [changes of T to A at positions 74 and 393]) to disrupt Fbw7-dependent regulation of cyclin E, its prototypic substrate, and to examine the consequences of cyclin E dysregulation for HSC function. Serial transplantation revealed that cyclin E^{T74A T393A} HSCs self-renewed normally; however, we identified defects in their multilineage reconstituting capacity. By inducing hematologic stress, we exposed an impaired self-renewal phenotype in cyclin E knock-in HSCs that was associated with defective cell cycle exit and the emergence of chromosome instability (CIN). Importantly, p53 deletion induced both defects in self-renewal and multilineage reconstitution in cyclin E knock-in HSCs with serial transplantation and CIN in hematopoietic stem and progenitor cells. Moreover, CIN was a feature of fatal T-cell malignancies that ultimately developed in recipients of cyclin E^{T74A T393A}; p53-null HSCs. Together, our findings demonstrate the importance of Fbw7-dependent cyclin E control to the hematopoietic system and highlight CIN as a characteristic feature of HSC dysfunction and malignancy induced by deregulated cyclin E.     

Impaired Humoral Immunity and Tolerance in K14-VEGFR-3-Ig Mice That Lack Dermal Lymphatic Drainage

Lymphatic vessels transport interstitial fluid, soluble Ag, and immune cells from peripheral tissues to lymph nodes (LNs), yet the contribution of peripheral lymphatic drainage to adaptive immunity remains poorly understood. We examined immune responses to dermal vaccination and contact hypersensitivity (CHS) challenge in K14-VEGFR-3-Ig mice, which lack dermal lymphatic capillaries and experience markedly depressed transport of solutes and dendritic cells from the skin to draining LNs. In response to dermal immunization, K14-VEGFR-3-Ig mice produced lower Ab titers. In contrast, although delayed, T cell responses were robust after 21 d, including high levels of Ag-specific CD8(+) T cells and production of IFN-γ, IL-4, and IL-10 upon restimulation. T cell-mediated CHS responses were strong in K14-VEGFR-3-Ig mice, but importantly, their ability to induce CHS tolerance in the skin was impaired. In addition, 1-y-old mice displayed multiple signs of autoimmunity. These data suggest that lymphatic drainage plays more important roles in regulating humoral immunity and peripheral tolerance than in effector T cell immunity.     

A Mutation in Vicia faba Results in Ineffective Nodules with Impaired Bacteroid Differentiation and Reduced Synthesis of Late Nodulins

Although numerous reports have documented the effect of bacterially-inducedineffectiveness on root nodule structure, function, and plantgene expression, few studies have detailed the effect of theplant genome on similar parameters. In this report effective(N₂-fixing) broadbean {Vicia faba L.) and plant-controlled ineffective(non-N₂-fixing) broadbean recessive for the sym-1 gene werecompared for nodule structure, developmental expression of noduleenzyme activities, enzyme proteins, and mRNAs involved in Nassimilation, leghemoglobin (Lb) synthesis, and acetylene reductionactivity (ARA). During development of effective wild-type nodules,glutamine synthetase (GS), aspartate aminotransferase (AAT),phosphoenolpyruvate carboxylase (PEPC) and NADH-glutamate synthase(GOGAT) activities and enzyme proteins increased coincidentwith nodule ARA. The increases in GS, AAT, and PEPC were associatedwith increased synthesis of mRNAs for these proteins. Synthesisof Lb polypeptides and mRNAs during development of effectivenodules was similar to that of GS, AAT, and PEPC. By contrast,ineffective sym-1 nodules displayed little or no ARA and hadneither the increases in enzyme activities nor enzyme proteinsand mRNAs as seen for effective nodules. The effect of the sym-1gene appeared to occur late in nodule development at eitherthe stage of bacterial release from infection threads or differentiationof bacteria into bacteroids. High in vitro enzyme activities,enzyme polypeptides, and mRNA levels in parental effective noduleswere dependent upon a signal associated with effective bacteroidsthat was lacking in sym-1 nodules. Nodule organogenesis didnot appear to be a signal for the induction of GS, PEPC, AAT,and Lb expression in sym-1 nodules. Key words: Vicia faba, mutation, sym-1 gene, nodules