JAK/STAT/PKCδ molecular pathways in synovial fluid T lymphocytes reflect the in vivo T helper-17 expansion in psoriatic arthritis

Looking to the sustained psoriatic arthritis (PsA) joint as a model of local human inflammation, this study was designed to assess the T lymphocyte signal transduction pathways potentially involved in this chronic immune-mediated inflammatory process, as characterized by direct ex vivo analysis of T helper (Th)-17 T effector (Teff) cell phenotypes in synovial fluid (SF) and peripheral blood (PB) of clinically active PsA patients. The reverse-phase protein arrays (RPPA) technique was employed to identify STAT3, STAT1, JAK1, JAK2, PKCδ and ERK1/2 phosphoprotein levels on total T cell lysates in SF samples of PsA patients. Frequencies of T CD4⁺IL-17A-F⁺ and T CD4⁺IL-23R⁺ Th17 cells were quantified in SF and matched PB of PsA patients by flow cytometry and compared with PB of healthy controls (HC). Increased levels of JAK1, STAT3, STAT1 and PKCδ phosphoproteins were found in SF T cells of PsA patients, compared with PB of HC. The expansion of T CD4⁺IL-17A-F⁺ cells, as well as of T CD4⁺ cells expressing IL-23Rp19 (T CD4⁺ IL-23R⁺), considered as the pathogenic phenotype of effector Th17 cells, was found to be confined to the joints of PsA patients, as the frequencies of both populations were significantly higher in SF than in matched PB, or in PB of HC. In conclusion, T lymphocyte signal transduction pathway mapping revealed an enhanced activation of JAK1/STAT3/STAT1 and PKCδ phosphoproteins that may drive the local inflammatory process, characterized by the in vivo expansion of T CD4⁺IL-17A-F⁺ and T CD4⁺IL-23R⁺ Th17 Teff cells in SF of clinically active joints of PsA patients.     

Osmotic Fragility and Erythrocyte Size in Iguana iguana (Sauria – Iguanidae) in Captivity

The erythrocyte size and osmotic fragility were studied in blood samples from adult (n= 40) and juvenile (n= 40) specimens of Iguana iguana. In fresh preparations the erythrocytes were large, oval cells. The largest diameters were 17.06 ± 2.5 mm (juvenile) and 16.20 ± 1.25 mm (adults), and the smallest diameters were 8.23 ± 1.87 mm (juvenile) and 9.00 ± 1.00 mm (adults). In fixed and stained preparations, the largest erythrocyte diameters were 15.28 ± 3.3 mm (juvenile) and 15.51 ± 1.3 mm (adults), and the smallest were 7.82 ± 0.65 mm (juvenile) and 7.85 ± 0.8 mm (adults). The haematocrit value for both juvenile and adult specimens was 27 ± 2%; the red blood cell counts were 1.3 ± 0.43×10¹²/l (juvenile) and 1.2 ± 0.35×10¹²/l (adults). Although no significant differences were observed in the cumulative osmotic curves, the derivative curve of adult specimens indicates the presence of at least two erythrocyte populations with osmotic fragilities at about 70 and 60 mm NaCI, representing 27% and 73% of the total cells, respectively. In samples from juvenile specimens, a major peak at about 70 mm NaCI was observed, which represented 85% of the total cell population. The difference in osmotic resistance between these erythrocyte subpopulations is correlated with their respective geometrical parameters, and compared to that of erythrocytes from other vertebrates.     

Characterization of IncA/C conjugative plasmid harboring blaTEM-52 and blaCTX-M-15 extended-spectrum β-lactamases in clinical isolates of Escherichia coli in Tunisia

To characterize the extended-spectrum β-lactamases (ESBLs) as well as their genetic environment in different isolates of Escherichia coli from patients with repeated urinary tract infections, large multidrug resistance (MDR) plasmids have been found. Definitive evidence for the presence of an A/C incompatibility complex (IncA/C) plasmid in the MDR isolates was provided by the probing of plasmids extracted from the clinical isolates. Conjugation experiments showed that bla genes were transferred by conjugation from the ten E. coli clinical isolates to E. coli XL1-Blue recipient. A comparative restriction fragment length polymorphism (RFLP) analysis of these plasmids showed that they are genetically similar, while the overall similarity of these plasmids supports the likelihood of recent movements among these E. coli isolates. Polymerase chain reaction (PCR) amplification and sequencing of the amplicons showed that the IncA/C plasmids harbor two ESBLs, identified as TEM-52 and CTX-M-15. Analysis of the plasmid DNA surrounding the bla _CTX-M-15 gene in the clinical isolates under study revealed a partially truncated fragment of ISEcp1 tnpA transposase. This result indicates the variety of genetic events that have enabled associations between ISEcp1 sequences and bla _CTX-M-15 genes in these clinical isolates.     

Regulation of hemagglutinin/protease expression by the VarS/VarA–CsrA/B/C/D system in Vibrio cholerae

In this study, through the analysis of Vibrio cholerae 2740-80 mutant strains produced by the cholera toxin subunit B gene containing Mariner-based transposon, we found that disruption of the varS gene, a member of the recently reported sensory system VarS/VarA–CsrA/B/C/D, resulted in altered expression of hemagglutinin/protease A. To further investigate the connection between VarS and HapA, we generated an additional varS mutant, V. cholerae 2740-80-VS, and examined the effect of this mutation on expression of HapA and of genes in the VarS/VarA–CsrA/B/C/D system. 2740-80-VS showed decreased expression of varS, csrB/C, hapR, and hapA along with increased biofilm production. Interestingly, expression of the alternative sigma factor σ^s, which is important for adaptation to environmental stress, was also decreased in this mutant. These results indicate that the VarS/VarA–CsrA/B/C/D system is involved in the control of HapA expression and biofilm production in V. cholerae 2740-80 through HapR regulation, and also that VarS/VarA controls expression of σ^s for HapA regulation.     

β2 Integrin/ICAM Expression in Crohn's Disease

We have previously reported on the expression of the β2 integrin family of adhesion molecules and their ligands, the ICAM molecules, in the normal human intestine. These molecules likely have a role to play in the inflammatory response and, therefore, were studied in a group of patients with Crohn's disease. A comprehensive study was undertaken in both colon (n= 8) and ileum (n= 10) specimens from 15 patients who underwent surgical resections. Immunohistochemistry was performed for CD18, CD11a, CD11b, CD11c, αd, ICAM-1, ICAM-2, and ICAM-3. Each of the mucosal, submucosal, muscle, and adventitial layers were scored for expression. Specimens from normal colon (n= 15), normal ileum (n= 6), and ulcerative colitis (n= 7) were used for comparisons. Compared with normal, the expression in the colon mucosa and submucosa in Crohn's disease was increased for all β2 integrins. Mucosal CD11c expression was significantly greater in Crohn's disease than in ulcerative colitis. In the colon muscle and adventitial layers the expression in Crohn's disease was similar to normal but increased compared with ulcerative colitis. In Crohn's disease ileum, the β2 integrin mucosal and submucosal expression was similar to normal; however, muscle and adventitial expression was increased, particularly for CD11c. Colon ICAM-1, ICAM-2, and ICAM-3 expression in Crohn's disease was similar to that seen in ulcerative colitis. ICAM-1 was predominantly expressed on endothelium but in the inflammatory bowel diseases was also evident on mucosal mononuclear cells. ICAM-1 and ICAM-2 expression was increased in Crohn's disease colon and ileum compared with normals. This was most notable in ileal mucosa since ICAM-2 is typically absent in normal ileal mucosa. In summary, we are reporting a comprehensive immunohistochemical study of the differential expression of β2 integrins, including the newly described αd molecule, and the ICAM molecules in all layers of the colon and ileum from patients with Crohn's disease. The increased expression of these molecules may have implications for therapeutic interventions in Crohn's disease.     

Natural Infections of Angiostrongylus vasorum and Crenosoma vulpis in Dogs in Germany (2007–2009)

In order to assess the occurrence and regional geographical distribution of Angiostrongylus vasorum and Crenosoma vulpis in Germany, faecal samples of 810 dogs with clinical symptoms of respiratory and circulatory disease, bleeding disorder and/or neurological signs were collected from September 2007 to March 2009. The zinc chloride/sodium chloride flotation and Baermann funnel technique were used to examine the samples for presence of lungworm larvae. Infections with lungworms were diagnosed in 105 (13.0%) of the examined dogs. A. vasorum and C. vulpis were found in 60 (7.4%) and 49 (6.0%) faecal samples, respectively. 33 A. vasorum- and 12 C. vulpis-positive dogs were located in Baden-Württemberg, 13 and 12 in North Rhine-Westphalia, 3 and 4 in Bavaria, 1 and 7 in Rhineland-Palatinate, 7 and 4 in Saarland, 1 and 2 in Saxony, respectively. In Brandenburg only 2 dogs with A. vasorum and in Hesse a total of 5 dogs with C. vulpis were detected. In Mecklenburg-Western Pomerania, Lower Saxony and Thuringia only 1 dog with C. vulpis was detected at a time. 4 dogs were coinfected with A. vasorum and C. vulpis. These surprisingly high prevalence rates indicate that both parasites are endemic in Germany.     

Neuromediators in inflammation—a macrophage/nerve connection

Macrophages are key players not only during initiation of inflammation but also during its resolution. This is achieved by their high functional plasticity and the demand to recognize an enormous repertoire of danger signals and cytokines/chemokines derived from adaptive immune cells. Studies predominantly conducted over the last two decades implicate that macrophage responses are also modulated by neuronal mediators such as neurotransmitters or neurotrophic factors. Here we summarize the current understanding of neuromediator-dependent interplay between macrophages and the nervous system.     

IL-24/MDA-7 Suppressed the Transplantation Tumor in BALB/c Mice

It is well-documented that interleukin-24 （IL-24） can induce apoptosis in a large spectrum of human cancer derived cell lines, but the effect of MDA-7/IL-24 gene transfer on mouse melanoma cells remains unknown. The eukaryotic expressing plasmid of IL-24 （pEGFP-IL-24） was constructed by DNA recombination technique. The recombination plasmid and empty vector were transfected into B16F0 cells and the expressions of IL-24 were determined by LSM, the proliferation of B16F0 cells was measured by MTT assay, and apoptosis rate and cell-cycle distribution of B16F0 cells were measured by FCM. The inhibitory effect of IL-24 gene transfection in mouse solid tumor was observed and measured. Compared with the control, the proliferation of B16F0 cells was inhibited by transfection with pEGFP-IL-24 and the G2/M phase of the transfected cells was also increased. Moreover, the percentage of mice with detectable tumor was decreased after inoculated with B16F0 cells transfected with pEGFP-IL-24. Growth rate of tumor in mouse model was significantly inhibited in IL-24 gene therapy group compared with the control. Proliferation of B16F0 cells was inhibited by pEGFP-IL-24 transfection. The intratumor injection of pEGFP-IL-24 could inhibit the growth of solid tumor in mice remarkably. Cellular ＆ Molecular Immunology.     

The role of integrin-β/FAK in cyclic mechanical stimulation in MG-63 cells

Objective: This study aims to explore the function of Integrin-β/FAK in the mechanical signal transduction and the connection with downstream ERK signal pathways. Methods: Human osteosarcoma MG63 cell lines were used in this study. The effects of mechanical strain on the Integrin-β₁ expression, FAK and ERK signal pathway in Human osteosarcoma MG63 cells were detected using RT-PCR and Western-blotting methods. The localization of FAK in Human osteosarcoma MG63 cells were determined using immunofluorescent method. The interaction between Integrin-β₁ and FAK were detected by using co-immunoprecipitation method. Results: The expression of Integrin-β₁ shows a notable bimodel distribution, mechanical strain stimulation can promote Integrin-β₁ expression and the phosphorylation of FAK and ERK, mechanical strain activated FAK and ERK mediated by Integrin-β₁. Conclusion: Integrin-β₁ may play an important role in osteoblast proliferation differentiation process, it might feel external strain stimulation through ECM composition and makes FAK phosphated through the interaction with FAK, thus causing a series of activation of signal molecules. Finally it reduces MAPK (ERK) activation and cellular responses to finish mechanical signal transduction.     

Does Depression Screening in Schools Reduce Adolescent Racial/Ethnic Disparities in Accessing Treatment?

Although placing mental health services in schools increases access to care, racial/ethnic disparities persist within the scope of school-based mental health services. Universal mental health screening is a potential strategy to increase problem detection and reduce disparities in care provision. However, no study has experimentally tested the effect of universal screening on patterns of service utilization across racial groups and the potential to reduce disparities. Using a cluster randomized design, we compared service linkage patterns among 7th- and 8th-grade Asian American and Latino students (N = 2,494; M_age = 13.65) in schools that either conducted or did not conduct universal depression screening. Multilevel analyses showed that enrollment in a universal screening school, Latino ethnicity, and low academic performance were associated with greater likelihood of referral. However, these factors were not related to caregiver consent or treatment initiation. Screening-triggered referrals were less likely to result in caregiver consent compared to routine referrals. Furthermore, universal screening did not result in a statistically significant reduction in racial/ethnic disparities in treatment referral. Implications for engaging ethnic minority families beyond the point of problem recognition and referral are discussed.     

Trends in Underweight and Overweight/Obesity Prevalence in Chinese Youth, 2004–2009

Background

There is a paucity of recent data on Chinese childhood overweight and underweight prevalence especially since 2004.

Purpose

The purpose of this study was to examine trends in underweight and overweight/obesity (“overweight” hereafter) prevalence and energy balance-related behaviors of Chinese youth from 2004 to 2009.

Methods

Data from the China Health and Nutrition Survey, 2004–2009 (N?=?4,061 students aged 6–18 years), were analyzed. Trained health workers took anthropometric measures at the participant’s house or at a local clinic following a reference protocol recommended by the World Health Organization. The international age- and sex-specific body mass index reference standard proposed by the International Obesity Task Force was used to define underweight and overweight children in this study.

Results

Among 6- to 11-year-old boys, underweight prevalence increased from 14.5 % (2004) to 20.1 % (2009, p?=?0.068). Among 12- to 18-year-old boys, however, overweight prevalence increased from 7.5 to 12.6 % (p?=?0.034). From 2004 to 2009, after-school sedentary behavior increased from 2.3 to 3.4 h/day for 6- to 11-year-olds (p?<?0.001) and from 2.2 to 3.1 h/day for 12- to 18-year-olds (p?<?0.01). Meanwhile, the total energy intake decreased 7 % for 6- to 11-year-olds (p?<?0.05) and 10 % for 12- to 18-year-olds (p?<?0.01).

Conclusions

Both underweight and overweight Chinese students are increasing, with underweight increases more pronounced in 6- to 11-year-olds and overweight increases more pronounced in 12- to 18-year-olds. Nationwide efficacious interventions are needed that improve the diet, decrease sedentary behavior, and encourage a healthy and realistic body image in Chinese youth.     

The pathogenic role of cystathionine γ-lyase/hydrogen sulfide in streptozotocin-induced diabetes in mice

Reduced β-cell mass and increased activities of ATP-sensitive K(+) channels in pancreatic β cells are associated with the pathogenesis of diabetes. Cystathionine γ-lyase (CSE) is a major hydrogen sulfide (H(2)S)-producing enzyme in pancreatic β cells. Herein, we examine the effects of genetic and pharmacologic ablation of CSE on β-cell functions and their correlation with streptozotocin (STZ)-induced diabetes. Compared with wild-type mice, CSE knockout (CSE KO) mice that received STZ injections exhibited a delayed onset of diabetic status. The application of dl-propargylglycine (PPG) to inhibit CSE activity protected wild-type mice from STZ-induced hyperglycemia and hypoinsulinemia. STZ significantly increased pancreatic H(2)S production in wild-type mice but not in CSE KO mice. STZ induced more apoptotic β-cell death in wild-type mice than in CSE KO mice. STZ exposure decreased the viability of cultured INS-1E cells, which was partly reversed by PPG co-treatment. STZ also significantly stimulated H(2)S production in cultured INS-1E cells. In addition, STZ stimulated ATP-sensitive K(+) currents in pancreatic β cells from wild-type mice but not in the presence of PPG or in β cells from CSE KO mice. Sodium hydrosulfide injection instantly increased blood glucose, decreased plasma insulin, and deteriorated glucose tolerance in mice. Take together, these results provide evidence that the CSE/H(2)S system plays a critical role in regulating β-cell functions.     

TH1/TH2 paradigm in pregnancy: paradigm lost? Cytokines in pregnancy/early abortion: reexamining the TH1/TH2 paradigm

In this paper, we briefly survey the history of concepts in reproductive immunology from antibody-mediated tolerance to the "fetal allograft" to the current concept of an embryo "bathing in a sea of cytokines". We then review the paradigm that "allopregnancy is a Th2 phenomenon" and some of the evidence gained in animals and humans supporting it. We continue by discussing the light it sheds on immunologically caused recurrent abortion, and the present status of the concepts. We next show the limits of the Th1/Th2 paradigm by reviewing the role of "inflammatory" cytokines in implantation (as first seen with leukemia inhibitory factor). We go on to discuss recent data showing that interferon-gamma is not solely a "bad guy", e.g. abortifacient as the paradigm would predict, but is needed at low doses for the vascular development and transformation of uterine spiral arteries required for implantation and successful pregnancy. We conclude by discussing the emerging role of NK and IL-12, IL-15, IL-18 tripods and other cytokines in local angiogenesis and tissue remodelling, a series of new data bringing us well beyond the Th1/Th2 paradigm in pregnancy which, in this context, appears now obsolete and an oversimplification, although it has indeed been useful at first. Rather, step-specific events have to be considered and a key role is seen in local tissue remodelling, in which immune cytokines play an important role while not always being secreted by immune cells.     

Human T-lymphoid progenitors generated in a feeder-cell-free Delta-like-4 culture system promote T-cell reconstitution in NOD/SCID/γc(-/-) mice

Slow T-cell reconstitution is a major clinical concern after transplantation of cord blood (CB)-derived hematopoietic stem cells. Adoptive transfer of in vitro-generated T-cell progenitors has emerged as a promising strategy for promoting de novo thymopoiesis and thus accelerating T-cell reconstitution. Here, we describe the development of a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Culture of human CD34(+) CB cells in this new DL-4 system enabled the in vitro generation of large amounts of T-cell progenitor cells that (a) displayed the phenotypic and molecular signatures of early thymic progenitors and (b) had high T lymphopoietic potential. When transferred into NOD/SCID/γc(-/-) (NSG) mice, DL-4 primed T-cell progenitors migrated to the thymus and developed into functional, mature, polyclonal αβ T cells that subsequently left the thymus and accelerated T-cell reconstitution. T-cell reconstitution was even faster and more robust when ex vivo-manipulated and nonmanipulated CB samples were simultaneously injected into NSG mice (i.e., a situation reminiscent of the double CB transplant setting). This work provides further evidence of the ability of in vitro-generated human T-cell progenitors to accelerate T-cell reconstitution and also introduces a feeder-cell-free culture technique with the potential for rapid, safe transfer to a clinical setting.     

Testing for HER-2/neu in breast cancer: is fluorescence in situ hybridization superior in predicting outcome?

Testing for alterations in HER-2/neu in breast cancer has become increasingly popular in recent years, particularly with the recent development of a humanized antiHER-2/neu monoclonal antibody, trastuzumab, which is currently being employed in conjunction with cytotoxic chemotherapy to treat metastatic breast cancer in patients whose tumors exhibit this HER-2/neu alteration. Controversy exists not only on the optimal method of laboratory testing for this HER-2/neu alteration (i.e., fluorescence in situ hybridization (FISH) versus immunohistochemistry (IHC) versus others), but also on the type of reagents used for a given method. A plethora of published studies on tissue-based HER-2/neu testing has recently appeared in many peer-reviewed journals; many have concluded that IHC could be used as a first-line screening test, with the recommendation of FISH to confirm indeterminate results. In contrast to these studies, a recent study by Pauletti et al. showed that HER-2/neu testing by IHC does not predict clinical outcome as accurately as does FISH. This commentary discusses the findings of this study, within a broader review of critical issues relating to HER-2/neu testing in breast cancer.     

Intracellular pH regulation in cultured rat astrocytes in CO2/HCO 3 − -containing media

We studied the regulation of intracellular pH (pH_i) and the mechanisms of pH_i regulation in cultured rat astrocytes using microspectrofluorometry and the pH-sensitive fluorophore 2,7-bis(carboxyethyl-)-5,6-carboxyfluorescein. Control pH_i was 7.00±0.02 in HCO ₃ ^- containing solutions at an extracellular pH of 7.35. Addition of 4, 4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) or amiloride decreased pH_i, as did removal of extracellular Na⁺, while removal of extracellular Cl^- was followed by an increase in pH_i. Following exposure to an acid transient induced by increasing the CO₂ content from 5 to 15%, pH_i rapidly returned to base line, with an average initial rate of recovery of 0.10 pH units min^-1 (corresponding to a mean acid extrusion rate of 6.3±0.36 mmolo 1^-1 min^-1). Regulation of pH_i was impaired when either amiloride or DIDS was added or Cl^- was removed. This inhibition was enhanced when both DIDS and amiloride were present, and pH_i regulation was completely blocked in the absence of extracellular Na⁺. The rapid regulation of pH_i normally seen following a transient alkalinisation was not inhibited by amiloride or removal of Na⁺, but was partially inhibited by DIDS and by the absence of extracellular Cl^-. The results are compatible with the presence of at least three different pH_i-regulating mechanisms: a Na⁺/H⁺ antiporter, a Na⁺-dependent HCO ₃ ^- /Cl^- exchanger (both regulating pH_i during a transient acidification), and a passive Cl^-/HCO ₃ ^- exchanger (regulating pH_i during transient alkalinisation). The results fail to provide firm evidence of the presence of an electrogenic Na⁺/HCO ₃ ^- symporter.     

Ischemia/reperfusion-induced changes in intracellular free Ca2+ levels in rat skeletal muscle fibers – an in vivo study

Accumulation of intracellular free calcium (Ca2+i) may play an essential role in the ischemia/reperfusion injury of skeletal muscle. Although it has been shown that Ca2+i levels significantly increase during ischemia/reperfusion, it is still a matter of debate whether Ca2+i increases during ischemia alone. It was the aim of this study to monitor the in vivo Ca2+i levels in the rat spinotrapezius muscle during ischemia of varying duration and reperfusion, using a ratiometric fluorescence technique, and to investigate the relationship between the postischemic flow patterns and Ca2+i, if any. The muscle was loaded with Indo-1/AM and imaged by a cooled digital camera. Pre- and postischemic tissue perfusion was assessed by means of an analogue camera. Our results show that short-term ischemia (5, 15 and 30 min) and subsequent reperfusion (60 min) does not alter Ca2+i homeostasis and that tissue perfusion promptly recovers after the insult. One or two hours of ischemia resulted in changes in Ca2+i levels, varying from preparation to preparation; increases in some and no changes in others. In these preparations three distinct flow patterns - normal, compromised and no-reflow - could be distinguished during the 60-min reperfusion. Our main conclusion is that in skeletal muscle Ca2+i levels may increase, the increase probably depending on the muscle fiber type exposed.