Alterations in immune cell subsets and their cytokine secretion profile in childhood idiopathic thrombocytopenic purpura (ITP)

Immune thrombocytopenic purpura (ITP) is acquired autoimmune disease in children characterized by the breakdown of immune tolerance. This work is designed to explore the contribution of different lymphocyte subsets in acute and chronic ITP children. Imbalance in the T helper type 1 (Th1)/Th2 cytokine secretion profile was investigated. The frequency of T (CD3⁺, CD4⁺, CD8⁺) and B (CD19⁺) lymphocytes, natural killer (NK) (CD16⁺56⁺) and regulatory T (T_reg) [CD4⁺CD25^+highforkhead box protein 3 (FoxP3)⁺] cells was investigated by flow cytometry in 35 ITP children (15 acute and 20 chronic) and 10 healthy controls. Plasma levels of Th1 cytokines [interferon (IFN-γ) and tumour necrosis factor (TNF-α)] and Th2 [interleukin (IL)-4, IL-6 and IL-10)] cytokines were measured using enzyme-linked immunosorbent assay (ELISA). The percentage of T_reg (P < 0·001) and natural killer (NK) (P < 0·001) cells were significantly decreased in ITP patients compared to healthy controls. A negative correlation was reported between the percentage of T_reg cells and development of acute (r = −0·737; P < 0·01) and chronic (r = −0·515; P < 0·01) disease. All evaluated cytokines (IFN-γ, TNF-α, IL-4, IL-6 and IL-10) were elevated significantly in ITP patients (P < 0·001, P < 0·05, P < 0·05, P < 0·05 and P < 0·001, respectively) compared to controls. In conclusion, our data shed some light on the fundamental role of immune cells and their related cytokines in ITP patients. The loss of tolerance in ITP may contribute to the dysfunction of T_regs. Understanding the role of T cell subsets will permit a better control of autoimmunity through manipulation of their cytokine network.     

Improved emergency myelopoiesis and survival in neonatal sepsis by caspase‐1/11 ablation

Over one million newborns die annually from sepsis with the highest mortality in premature and low-birthweight infants. The inflammasome plays a central role in the regulation of innate immunity and inflammation, and is presumed to be involved in protective immunity, in large part through the caspase-1-dependent activation of interleukin-1β (IL-1β) and IL-18. Studies in endotoxic shock, however, suggest that endogenous caspase-1 activity and the inflammasome contribute to mortality primarily by promoting excessive systemic inflammatory responses. We examined whether caspase-1 and the inflammasome also regulate neonatal inflammation, host protective immunity and myelopoiesis during polymicrobial sepsis. Neonatal (5–7 days) C57BL/6 and caspase-1/11^−/− mice underwent a low-lethality caecal slurry model of intra-abdominal sepsis (LD_25–45). Ablation of caspase-1/11, but not apoptosis-associated speck-like protein containing a CARD domain or nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), improved neonatal survival following septic challenge compared with wild-type mice (P < 0·001), with decreased concentrations of inflammatory cytokines in the serum and peritoneum. Surprisingly, caspase-1/11^−/− neonates also exhibited increased bone marrow and splenic haematopoietic stem cell expansion (P < 0·001), and increased concentrations of granulocyte and macrophage colony-stimulating factors in the peritoneum (P < 0·001) after sepsis. Ablation of caspase-1/11 signalling was also associated with increased recruitment of peritoneal macrophages and neutrophils (P < 0·001), increased phagocytosis by neutrophils (P = 0·003), and decreased bacterial colonization (P = 0·02) in the peritoneum. These findings suggest that endogenous caspase-1/11 activity, independent of the NLRP3 inflammasome, not only promotes the magnitude of the inflammatory response, but also suppresses protective immunity in the neonate, so contributing to innate immune dysfunction and poor survival in neonatal sepsis.     

Significance of IL-1β and IL-1 receptor antagonist (IL-1Ra) in bronchoalveolar lavage fluid (BALF) in patients with diffuse panbronchiolitis (DPB)

We evaluated the effect of erythromycin therapy on pulmonary function tests and the airway inflammatory response of patients with DPB. The number of neutrophils in BALF obtained from DPB patients was significantly higher than that of healthy volunteers. Treatment with erythromycin (600 mg/day for 12·9 ± 9·5 months (mean ±s.d.)) significantly reduced the total number of cells and neutrophils in the airway, and significantly improved pulmonary function tests. The levels of IL-1β and IL-8 were significantly higher in DPB compared with healthy volunteers (P < 0·05, P < 0·05, respectively). IL-1 Ra in patients is considered to have a weak inhibitory activity for IL-1β, with approximately five-fold concentration of IL-1β compared with that in healthy volunteers (approx. nine-fold concentration of IL-1β). Erythromycin therapy significantly reduced these cytokines to levels comparable to those of healthy volunteers, and produced a trend toward reduction in the level of IL-1Ra in BALF. The level of IL-1β correlated significantly with the concentration of neutrophils in BALF (r = 0·72, P < 0·01), as well as with the level of IL-1Ra (r = 0·688, P < 0·05) and IL-8 (r = 0·653, P < 0·05). A nearly significant or significant correlation was observed between the concentration of neutrophils and levels of IL-1Ra or IL-8 in BALF (r = 0·526, P = 0·053 or r = 0·776, P < 0·01, respectively). There was also a significant relationship between FEV, and the concentration of neutrophils in BALF (r = 0·524, P < 0·05). Our results suggest that the relative amounts of IL-1β and IL-1Ra or IL-8 may contribute, at least in part, to the neutrophil-mediated chronic airway inflammation in patients with chronic airway disease, and long-term erythromycin therapy may down-regulate the vigorous cycle between the cytokine network and neutrophil accumulation, with resultant reduction of neutrophil-mediated inflammatory response.     

The relationship between thyroid function,serum monokine induced by interferon gamma and soluble interleukin‐2 receptor in thyroid autoimmune diseases

Interactions between cytokines play an important role in the development of thyroid autoimmunity. Using enzyme-linked immunosorbent assay we investigated serum concentrations of soluble interleukin-2 receptor (sIL-2R), interferon-gamma, tumour necrosis factor (TNF)-α, interleukin (IL)-10, CD30, monokine induced by interferon-gamma (MIG), cytotoxic T lymphocyte antigen-4 and markers of apoptosis decoy receptor 3 and Bcl-2 in 28 patients with hyperthyroid Graves'' disease (GD), 24 patients with untreated Hashimoto''s thyroiditis (HT) and 15 healthy controls. TNF-α, IL-10 and sIL-2R were higher in GD compared with HT and controls (TNF-α: 8·79 in GD versus 2·54 pg/ml in HT, P = 0·01; IL-10: 10·00 versus 3·10 versus 3·10 pg/ml, P₁ < 0·001, P₂ = 0·005; sIL-2R: 1·26 versus 0·64 versus 0·46 ng/ml, P < 0·001). MIG and CD30 were higher in HT compared with controls (649·22 ± 262·55 versus 312·95 ± 143·35 pg/ml, P = 0·037, 6·57 ± 2·35 versus 3·03 ± 1·04 U/ml, P = 0·036 respectively). In GD sIL-2R decreased when the euthyroid state was achieved (1·31 ± 0·64 versus 0·260 ± 0·11, n = 12, P < 0·001). sIL-2R correlated positively with free thyroxine (FT4) (R = 0·521, P = 0·000) and negatively with thyroid stimulating hormone (TSH) (R = −0·472, P = 0·00132). MIG correlated negatively with FT4 (R = −0·573, P = 0·00234) and positively with TSH (R = 0·462, P = 0·0179). The results suggest that serum concentrations of sIL-2R and MIG are related to thyroid function rather than to activation of autoimmunity.     

Combined Env- and Gag-specific T cell responses in relation to programmed death-1 receptor and CD4+ T cell loss rates in human immunodeficiency virus-1 infection

Additional progression markers for human immunodeficiency virus (HIV) infection are warranted. In this study we related antigen-specific responses in CD4⁺ and CD8⁺ T cells to CD38, reflecting chronic immune activation, and to CD4⁺ T cell loss rates. Clones transiently expressing CD107a (CD8⁺) or CD154 (CD4⁺) in response to Gag, Env and Nef overlapping peptide pools were identified, along with their expression of the inhibitory programmed death-1 receptor (PD-1) in fresh peripheral blood mononuclear cells (PBMC) from 31 patients off antiretroviral treatment (ART). HIV-specific CD8⁺ T cell responses dominated over CD4⁺ T cell responses, and among CD8⁺ responses, Gag and Nef responses were higher than Env-responses (P < 0·01). PD-1 on CD8⁺ HIV-specific subsets was higher than CMV-specific CD8⁺ cells (P < 0·01), whereas PD-1 on HIV-specific CD4⁺ cells was similar to PD-1 on CMV-specific CD4⁺ cells. Gag and Env CD8⁺ responses correlated oppositely to the CD4 loss rate. Env/Gag CD8⁺ response ratios, independently of PD-1 levels, correlated more strongly to CD4 change rates (r = −0·50 to −0·77, P < 0·01) than the total number of Gag-specific CD8⁺ cells (r = 0·44–0·85, P ≤ 0·02). The Env/Gag ratio performed better than CD38 and HIV-RNA in logistic regression analysis predicting CD4 change rate as a measure of progression. In conclusion, HIV-specific CD8⁺CD107a⁺ Env/Gag response ratio was a stronger predictor for progression than CD38 and HIV-RNA. The Env/Gag ratio may reflect the balance between possibly beneficial (Gag) and detrimental (Env) CD8⁺ T cell responses and should be explored further as a progression marker.     

Blood monocyte activation in rheumatoid arthritis: increased monocyte adhesiveness, integrin expression, and cytokine release

Infiltration of the synovium by mononuclear cells, namely lymphocytes and monocytes, is one of the main features of rheumatoid arthritis (RA) and is considered to be responsible for the development of the disease. In this study in 31 consecutive patients with RA, we investigated whether peripheral blood monocytes exhibited markers of cellular activation related to cell migration. Using flow cytometry with the respective specific antibodies, we studied the expression of integrins CD11a, CD11b, CD11c, CD49d (VLA-4), and CD49e (VLA-5) on monocytes from patients with RA and from normal (N) subjects. IL-1β, IL-6, and tumour necrosis factor-alpha (TNF-α) production by cultured monocytes was measured by immunoassay. Adhesiveness of monocytes was studied on various surfaces (plastic, human fibronectin, gelatin-coated plasma, subendothelial matrix) and on cultured endothelial cells under basal conditions or after stimulation by IL-1β. An increased number of CD14⁺ monocytes (Mo) from RA patients expressed the CD11b molecule (RA Mo = 90·3%, N Mo = 83·4%, P < 0·005). The expression of CD11b on CD14⁺ monocytes was significantly increased in RA patients (median fluorescence intensity (FI): RA Mo = 145 (range 80–466) units; normal Mo = 95 (range 24–164) units; P < 0·003). Production of extracellular IL-1β and IL-6 by RA monocytes was significantly enhanced compared with monocytes from normal subjects (IL-1β: RA = 2·65 ± 0·91 ng/ml versusN = 1·35 ± 0·85 pg/ml, P < 0·05; IL-6: RA = 4·83 ± 0·90 ng/ml versusN = 2·40 ± 0·95 ng/ml, P < 0·05). Compared with normal monocytes, RA monocytes exhibited increased adhesion to the various surfaces studied (plastic, P < 0·01; fibronectin, P < 0·01; and gelatin-coated normal or RA plasma, P < 0·01) as well as to unstimulated (P < 0·01) and IL-1β-stimulated endothelial cells (IL-1β for 4 h, P < 0·05; IL-1β for 24 h, P < 0·05). In our study, blood monocytes from RA patients exhibited features of activation related to cell adhesion.     

Tumour necrosis factor‐α plus interleukin‐10 low producer phenotype predicts acute kidney injury and death in intensive care unit patients

Genetic polymorphism studies of cytokines may provide an insight into the understanding of acute kidney injury (AKI) and death in intensive care unit (ICU) patients. The aim of this study was to investigate whether the genetic polymorphisms of −308 G < A tumour necrosis factor (TNF)-α, −174 G > C interleukin (IL)-6 and −1082 G > A IL-10 may predispose ICU patients to the development of AKI and/or death. In a prospective nested case–control study, 303 ICU patients and 244 healthy individuals were evaluated. The study group included ICU patients who developed AKI (n = 139) and 164 ICU patients without AKI. The GG genotype of TNF-α (low producer phenotype) was significantly lower in the with AKI than without AKI groups and healthy individuals (55 versus 62 versus 73%, respectively; P = 0·01). When genotypes were stratified into four categories of TNF-α/IL-10 combinations, it was observed that low TNF-α plus low IL-10 producer phenotypes were more prevalent in patients with AKI, renal replacement therapy and death (P < 0·05). In logistic regression analysis, low TNF-α producer plus low IL-10 producer phenotypes remained as independent risk factors for AKI and/or death [odds ratio (OR) = 2·37, 95% confidence interval (CI): 1·16–4·84; P = 0·02] and for renal replacement therapy (RRT) and/or death (OR = 3·82, 95% CI: 1·19–12·23; P = 0·02). In this study, the combination of low TNF-α plus low IL-10 producer phenotypes was an independent risk factor to AKI and/or death and RRT and/or death in critically ill patients. Our results should be validated in a larger prospective study with long-term follow-up to emphasize the combination of these genotypes as potential risk factors to AKI in critically ill patients.     

Predictive Factors Associated with the Reversibility of Post-transplantation Diabetes Mellitus Following Liver Transplantation

Post-transplantation diabetes mellitus (PTDM) is reversible in a considerable number of patients. We examined the prevalence and predictive factors of transient PTDM following liver transplantation. Forty-two of 74 PTDM patients showed the clinical features of transient PTDM. Compared with the persistent PTDM patients, they were characterized by younger age at the time of transplantation (49±7 vs. 53±8 yr, P<0.05), longer time before the development of PTDM (44±59 vs. 13±20 days, P<0.05), lower rate of hepatitis c virus seropositivity (0.0 vs. 9.4%, P<0.05), and use of mycophenolate mofetil (59.5 vs. 28.1%, P<0.05). Among these risk factors, age at the time of transplantation is the single independent predictive factor associated with the reversibility of PTDM.     

Nitric oxide production by polymorphonuclear leucocytes in infected cystic fibrosis sputum consumes oxygen

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes (PMNs), which consume a major part of present molecular oxygen (O₂) due to production of superoxide (O₂⁻). In this study, we show that the PMNs also consume O₂ for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the infected endobronchial mucus. Fresh expectorated sputum samples (n = 28) from chronically infected CF patients (n = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases (NOS) with N^G-monomethyl-L-arginine (L-NMMA) resulted in reduced O₂ consumption (P < 0·0008, n = 8) and a lower fraction of cells with fluorescence from the NO-indicator 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) (P < 0·002, n = 8). PMNs stained with DAF-FM and the superoxide indicator hydroethidine (HE) and host cells with inducible NOS (iNOS) were identified in the sputum. In addition, the production of the stable end-products of NO in CF sputum was correlated with the concentration of PMNs; NO₃⁻ (P < 0·04, r = 0·66, n = 10) and NO₂⁻ (P< 0·006, r = 0·78, n = 11). The present study suggests that besides consumption of O₂ for production of reactive oxygen species, the PMNs in CF sputum also consume O₂ for production of NO.     

Gastric emptying after pickle-juice ingestion in rested, euhydrated humans

Context:

Small volumes of pickle juice (PJ) relieve muscle cramps within 85 seconds of ingestion without significantly affecting plasma variables. This effect may be neurologic rather than metabolic. Understanding PJ''s gastric emptying would help to strengthen this theory.

Objective:

To compare gastric emptying and plasma variables after PJ and deionized water (DIW) ingestion.

Design:

Crossover study.

Setting:

Laboratory.

Patients or Other Participants:

Ten men (age  =  25.4 ± 0.7 years, height  =  177.1 ± 1.6 cm, mass  =  78.1 ± 3.6 kg).

Intervention(s):

Rested, euhydrated, and eunatremic participants ingested 7 mL·kg⁻¹ body mass of PJ or DIW on separate days.

Main Outcome Measure(s):

Gastric volume was measured at 0, 5, 10, 20, and 30 minutes postingestion (using the phenol red dilution technique). Percentage changes in plasma volume and plasma sodium concentration were measured preingestion (−45 minutes) and at 5, 10, 20, and 30 minutes postingestion.

Results:

Initial gastric volume was 624.5 ± 27.4 mL for PJ and 659.5 ± 43.8 mL for DIW (P > .05). Both fluids began to empty within the first 5 minutes (volume emptied: PJ  =  219.2 ± 39.1 mL, DIW  =  305.0 ± 40.5 mL, P < .05). Participants who ingested PJ did not empty further after the first 5 minutes (P > .05), whereas in those who ingested DIW, gastric volume decreased to 111.6 ± 39.9 mL by 30 minutes (P < .05). The DIW group emptied faster than the PJ group between 20 and 30 minutes postingestion (P < .05). Within 5 minutes of PJ ingestion, plasma volume decreased 4.8% ± 1.6%, whereas plasma sodium concentration increased 1.6 ± 0.5 mmol·L⁻¹ (P < .05). Similar changes occurred after DIW ingestion. Calculated plasma sodium content was unchanged for both fluids (P > .05).

Conclusions:

The initial decrease in gastric volume with both fluids is likely attributable to gastric distension. Failure of the PJ group to empty afterward is likely due to PJ''s osmolality and acidity. Cardiovascular reflexes resulting from gastric distension are likely responsible for the plasma volume shift and rise in plasma sodium concentration despite nonsignificant changes in plasma sodium content. These data support our theory that PJ does not relieve cramps via a metabolic mechanism.     

Mechanisms of corticosteroid action on lymphocyte subpopulations. III. Differential effects of dexamethasone administration on subpopulations of effector cells mediating cellular cytotoxicity in man

The present study investigated the effect of dexamethasone (DEX) administration on different populations of mononuclear cells and neutrophils mediating antibody-dependent cellular cytotoxicity (ADCC) against different target cells.

Mononuclear cells (lymphocytes and monocytes) and neutrophils were obtained from twenty-seven normal volunteers at 0, 4, 24 and 48 hr after oral administration of 21 mg of DEX. ADCC was determined utilizing the following targets: human red blood cells (HRBC), Chang liver cells (Ch) and human heart cells (HHC). The predominant mononuclear effector in HRBC killing was shown to be a monocyte and in Ch and HHC killing, a K cell. As previously shown, DEX produced a profound monocytopenia and lymphocytopenia at 4 hr with a return of lymphocyte counts to normal and monocyte counts to supra-normal at 24 hr. At the point of maximal monocytopenia, monocyte-mediated HRBC killing decreased from a geometric mean of 14 to 4 lytic units per 10⁸ effector cells (P<0·05) and rebounded at 24 hr to a mean of 39 lytic units (P<0·02) with the rebound monocytosis. At the point of absolute lymphopenia (4 hr), there was a relative enrichment in the proportion of lymphocytes bearing an Fc receptor (K cells, P<0·01). Concomitant with this was an increase in ADCC against Ch and HHC from geometric means of 1121 to 7172 lytic units and 939 to 7354 lytic units (P<0·001) respectively. Thus, a major action of DEX administration on mononuclear ADCC was to differentially enrich or deplete different effector cells to and from the circulation, causing changes in cytotoxicity. Since the cytotoxicity paralleled the proportion of effector cells, the cells remaining in the circulation following DEX administration retained normal antibody-dependent cytotoxic capabilities.

Neutrophil-mediated ADCC against HRBC significantly increased at 4 hr from a geometric mean of 3785 to 20142 lytic units (P<0·02) concomitant with the blood neutrophilia and remained elevated for 72 hr. Neutrophil ADCC against Ch and HHC was minimal and was not affected by DEX administration.

This study demonstrates differential effects of corticosteroids on antibody-dependent cell-mediated cytotoxicity, reflecting changes in proportion of circulating effector cell populations. These changes depend on (a) the target cell employed, (b) the effector cell mediating cytotoxicity and (c) the duration of time since the last dose of corticosteroid.

     

Acute cytomegalovirus infection and the host immune response. II. Relationship of suppressed in vitro lymphocyte reactivity to bacterial recall antigens and mitogens with the development of cytomegalovirus-induced lymphocyte reactivity

Cellular immunity in cytomegalovirus (CMV) infection was studied in eighteen patients with CMV infection in the acute stage and thereafter. Peripheral blood lymphocyte reactivity (LR) to mitogens, bacterial recall antigens and CMV was tested sequentially in fourteen out of eighteen patients and once in four patients and sex and age-matched healthy controls. LR to a cocktail of bacterial recall antigens was impaired (P<0·01) during the acute phase of the CMV infection (<50 days after onset of illness symptoms). Despite a gradual improvement during the first (convalescent and post-convalescent, 50–250 days) and second (follow-up) period (>250 days) it still remained markedly impaired as opposed to LR of the controls (P<0·01 and P<0·02 respectively). In ten out of eighteen patients twelve superinfections were suspected clinically and two viral and eight bacterial infections documented during the observation period of 1 year after onset of CMV infection. In vitro LR to Con A and PWM appeared grossly disturbed as opposed to PHA-LR which was decreased only slightly in the acute phase. Although a gradual improvement of mitogenic LR was seen after the acute phase, PWM-LR remained decreased during the follow-up period as opposed to that of the controls (P<0·05). CMV-specific LR to heat-inactivated AD 169 strain virion antigens was generally negative or lowgrade initially but a positive CMV-LR test was obtained in all patients studied sequentially during or after the acute phase. To a certain extent, the appearance of positive CMV-LR was associated with improvement of LR to bacterial recall antigens and mitogens. In individual cases CMV-LR development apparently preceded the restoration of the generalized immunosuppression as reflected by LR tests with mitogens and bacterial recall antigens. It is concluded that CMV infection of adults causes a (relatively) longlasting cellular immunosuppression. Possible pathogenetic mechanisms are discussed. A relationship with increased sensitivity to infections of an opportune nature after CMV infection is suggested.     

Bead-size directed distribution of Pseudomonas aeruginosa results in distinct inflammatory response in a mouse model of chronic lung infection

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by biofilms, tolerant to antibiotics and host responses. Instead, immune responses contribute to the tissue damage. However, this may depend on localization of infection in the upper conductive or in the peripheral respiratory zone. To study this we produced two distinct sizes of small alginate beads (SB) and large beads (LB) containing P. aeruginosa. In total, 175 BALB/c mice were infected with either SB or LB. At day 1 the quantitative bacteriology was higher in the SB group compared to the LB group (P < 0·003). For all time-points smaller biofilms were identified by Alcian blue staining in the SB group (P < 0·003). Similarly, the area of the airways in which biofilms were identified were smaller (P < 0·0001). A shift from exclusively endobronchial to both parenchymal and endobronchial localization of inflammation from day 1 to days 2/3 (P < 0·05), as well as a faster resolution of inflammation at days 5/6, was observed in the SB group (P < 0·03). Finally, both the polymorphonuclear neutrophil leucocyte (PMN) mobilizer granulocyte colony-stimulating factor (G-CSF) and chemoattractant macrophage inflammatory protein-2 (MIP-2) were increased at day 1 in the SB group (P < 0·0001). In conclusion, we have established a model enabling studies of host responses in different pulmonary zones. An effective recognition of and a more pronounced host response to infection in the peripheral zones, indicating that increased lung damage was demonstrated. Therefore, treatment of the chronic P. aeruginosa lung infection should be directed primarily at the peripheral lung zone by combined intravenous and inhalation antibiotic treatment.     

Up-regulation of CC chemokine receptor 6 on tonsillar T cells and its induction by in vitro stimulation with α-streptococci in patients with pustulosis palmaris et plantaris

Pustulosis palmaris et plantaris (PPP) is a tonsil-related disease; tonsillectomy is somewhat effective in treating the condition. However, the aetiological association between the tonsils and PPP has not yet been elucidated fully. Recently, some chemokines and chemokine receptors, including CC chemokine receptor (CCR) 4, CCR6 and CX chemokine receptor (CXCR) 3, have been reported to play important roles in the development of psoriasis, a disease related closely to PPP. In this study, we found that CCR6 expression on both tonsillar and peripheral blood T cells was up-regulated more intensively in PPP patients than in non-PPP patients (P < 0·001 for both), but CCR4 and CXCR3 expressions were not. In vitro stimulation with α-streptococcal antigen enhanced CCR6 expression significantly on tonsillar T cells in PPP patients (P < 0·05), but this was not observed in non-PPP patients. The chemotactic response of tonsillar T cells to the CCR6 ligand CC chemokine ligand (CCL) 20 was significantly higher in PPP patients than in non-PPP patients (P < 0·05). The percentage of CCR6-positive peripheral blood T cells decreased after tonsillectomy in PPP patients (P < 0·01); this decrease correlated with an improvement of skin lesions (P < 0·05, r = −0·63). The numbers of CCR6-positive cells and the expression of CCL20 were increased significantly in pathological lesions compared with non-pathological lesions in PPP skin (P < 0·01, P < 0·05 respectively). These results suggest that a novel immune response to α-streptococci may enhance CCR6 expression on T cells in tonsils and that CCR6-positive T cells may move to peripheral blood circulation, resulting in recruitment to target skin lesions expressing CCL20 in PPP patients. This may be one of the key roles in pathogenesis of the tonsil-related disease PPP.     

Lung transplantation affects expression of the chemokine receptor type 4 on specific T cell subsets

Alloreactive T cells that infiltrate the graft after lung transplantation (LTx) play a role in chronic rejection. Chemokines such as thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and monocyte chemotactic protein-1 (MCP-1) are produced locally in the lung and attract T cells via chemokine receptor 4 (CCR4). In a TARC gradient, cells expressing CCR4⁺⁺ migrate more efficiently than CCR4⁺-expressing cells. In this study, we compared the CCR4 expression of T cells in blood from 20 lung transplant recipients to healthy controls. We then examined whether CCR4 expression is associated with the occurrence of chronic rejection. The CCR4⁺⁺ expression was decreased on CD4 T cells from LTx patients (P < 0·0001) when compared to healthy controls. The analysis of CD4 T cell subsets showed that this decrease was present on central memory, effector memory and terminally differentiated T cells (P = 0·0007, P < 0·0001 and P = 0·05, respectively), while a trend was found for naive CD4 T cells (P = 0·06). Also, the expression of CCR4⁺ on regulatory T cells (T_regs) was decreased in LTx patients when compared to healthy controls (P = 0·02). Interestingly, the CCR4⁺⁺ expression on CD4 effector memory T cells was decreased in patients developing chronic rejection sometimes more than a year before the clinical diagnosis when compared to patients who did not (P = 0·04). The analysis of CD8 T cell subsets only showed the CCR4⁺ expression to be increased significantly on effector memory and terminally differentiated CD8 T cells (P = 0·02, P = 0·03, respectively) in LTx patients, but no relation was found in chronic rejection. In conclusion, the expression of CCR4 on T cell subsets was altered after LTx and appears to be related to chronic rejection.     

Serum titres of anti-glutamic acid decarboxylase-65 and anti-IA-2 autoantibodies are associated with different immunoregulatory milieu in newly diagnosed type 1 diabetes patients

Several studies correlated genetic background and pancreatic islet-cell autoantibody status (type and number) in type 1A diabetes mellitus (T1AD), but there are no data evaluating the relationship among these markers with serum cytokines, regulatory T cells and β cell function. This characterization has a potential importance with regard to T1AD patients'' stratification and follow-up in therapeutic prevention. In this study we showed that peripheral sera cytokines [interleukin (IL)-12, IL-6, II-1β, tumour necrosis factor (TNF)-α, IL-10] and chemokines (CXCL10, CXCL8, CXCL9, CCL2) measured were significantly higher in newly diagnosed T1AD patients when compared to healthy controls (P < 0·001). Among T1AD, we found a positive correlation between CXCL10 and CCL-2 (r = 0·80; P = 0·000), IL-8 and TNF-α (r = 0·60; P = 0·000); IL-8 and IL-12 (r = 0·57; P = 0·001) and TNF-α and IL-12 (r = 0·93; P = 0·000). Glutamic acid decarboxylase-65 (GAD-65) autoantibodies (GADA) were associated negatively with CXCL10 (r = −0·45; P = 0·011) and CCL2 (r = −0·65; P = 0·000), while IA-2A showed a negative correlation with IL-10 (r = −0·38; P = 0·027). Human leucocyte antigen (HLA) DR3, DR4 or DR3/DR4 and PTPN22 polymorphism did not show any association with pancreatic islet cell antibodies or cytokines studied. In summary, our results revealed that T1AD have a proinflammatory cytokine profile compared to healthy controls and that IA-2A sera titres seem to be associated with a more inflammatory peripheral cytokine/chemokine profile than GADA. A confirmation of these data in the pre-T1AD phase could help to explain the mechanistic of the well-known role of IA-2A as a more specific marker of beta-cell damage than GADA during the natural history of T1AD.     

The uncertainty (validity and reliability) of three electrothermometers in therapeutic modality research

Context: Data from electrothermometers are used to determine therapeutic modality usage, but the value of experimental results is only as good as the data collected.Objective: To determine the reliability and validity of 3 electrothermometers from 2 manufacturers.Design: A 3 × 4 × 17 factorial with repeated measures on 2 factors. Independent variables were trial (1, 2, 3), thermometer (mercury thermometer, Iso-Thermex calibrated from −50°C to 50°C, Iso-Thermex calibrated from −20°C to 80°C, and Datalogger), and time (17).Setting: Human Performance Research Center.Intervention(s): Eighteen thermocouples were inserted through the wall of a foamed polystyrene cooler, and 6 were connected to each of the 3 electrothermometers. The cooler was positioned on a stir plate and filled with room-temperature water (18.4°C). A mercury thermometer was immersed into the water bath. Measurements of the water bath were taken every 10 seconds for three 3-minute trials.Main Outcome Measure(s): The temperature variability of 3 electrothermometers was taken from a calibrated mercury thermometer.Results: The Iso-Thermex electrothermometers did not differ statistically from each other in uncertainty (validity error ± reliability error = 0.06°C ± 0.03°C ± 0.03°C ± 0.02°C, P < .05), but both differed from the Datalogger (0.64°C ± 0.20°C, P < .05). The Datalogger temperature was consistently higher than the mercury thermometer temperature.Conclusions: The Iso-Thermex electrothermometers were more stable than the Datalogger, and values were within the published uncertainty (±0.1°C) when used with PT-6 thermocouples. The Datalogger we used had an uncertainty of measurement greater than that indicated in the user''s manual (∼±0.52°C). Uncertainty of ±0.84°C can significantly influence the interpretation of results when intramuscular temperature changes are usually less than 5°C.     

Curative Resection of Hepatocellualr Carcinoma Using Modified Glissonean Pedicle Transection versus the Pringle Maneuver: A Case Control Study

Objective: The Glissonean pedicle transection method of liver resection has been found to shorten operative time and minimize intraoperative bleeding during liver segmentectomy. We have compared the feasibility, effectiveness, and safety of the Glissonean pedicle transection method with the Pringle maneuver in patients undergoing selective curative resection of large hepatocellualr carcinoma (HCC).Methods: Eligible patients with large (> 5 cm) nodular HCC (n = 50) were assigned to undergo curative hepatectomy using the Glissonean pedicle transection method (n = 25) or the Pringle maneuver (n = 25). Partial interruption of the infrahepatic inferior vena cava was incorporated to further reduce bleeding from liver transection. The primary outcome measure was postoperative changes in liver function from baseline. Secondary outcomes included operating time, volume of intraoperative blood loss/transfusion, and time to resolution of ascites.Results: The two groups were comparable in age, sex, site and size of the liver tumor, segment or lobe intended to be resected, and liver function reserve, and the results were not significant statistically. All patients underwent successful major hepatectomies using the assigned method, with the extent of major hepatectomy comparable in the two groups (P = 0.832). The Glissonean approach was associated with shorter hepatic inflow interruption (30.0 ± 12.0 min vs. 45.0 ± 13.0 min, P < 0.001), lower volume of blood loss (145.0 ± 20.0 mL vs. 298.0 ± 109.0 mL, P < 0.001), reduced requirement for transfusion (0.0 ± 0.0 mL vs. 200.0 ± 109.0 mL, P < 0.0001), and more rapid resolution of ascites (9.5 ± 1.2 d vs. 15.3 ± 2.4 d, P < 0.001). Postoperative liver function measures were comparable in the two groups, and the results were not significant statistically.Conclusion: The Glissonean pedicle transection method is a feasible, effective, and safe technique for hepatic inflow control during the curative resection of large nodular HCCs.     

Curcumin alleviates immune‐complex‐mediated glomerulonephritis in factor‐H‐deficient mice

Complement factor H (Cfh) is a key regulator of the complement cascade and protects C57BL/6 mice from immune complex-mediated complement-dependent glomerulonephritis. In chronic serum sickness (CSS) there are increased deposits of immune complexes in the glomeruli with inflammation and a scarring phenotype. As cucurmin is an effective anti-inflammatory agent and reduces complement activation, we hypothesized that it should alleviate renal disease in this setting. To determine the effectiveness of curcumin, an apoferritin-induced CSS model in Cfh-deficient (Cfh^−/−) mice was used. Curcumin treatment (30 mg/kg) given every day in parallel with apoferritin reduced glomerulonephritis and enhanced kidney function (blood urea nitrogen, 45·4 ± 7·5 versus 35·6 ± 5·1; albuminuria, 50·1 ± 7·1 versus 15·7 ± 7·1; glomerulonephritis, 2·62 + 0·25 versus 2 + 0·3, P < 0·05). In line with reduced IgG deposits in mice with CSS given curcumin, C9 deposits were reduced indicating reduced complement activation. Mice treated with curcumin had a significant reduction in the number of splenic CD19⁺ B cells and the ratio of CD19 : CD3 cells (P < 0·05) with no change in the T-cell population. Myeloperoxidase assay showed reduced macrophages in the kidney. However, a significant reduction in the M2 subset of splenic macrophages by apoferritin was prevented by curcumin, suggesting a protective function. Curcumin treatment reduced mRNA expression of inflammatory proteins monocyte chemoattractant protein-1 and transforming growth factor-β and matrix proteins, fibronectin, laminin and collagen. Our results clearly illustrate that curcumin reduces glomerulosclerosis, improves kidney function and could serve as a therapeutic agent during serum sickness.