Chronic obstructive pulmonary disease (COPD) and chronic heart failure (CHF) may coexist in elderly patients with a history of smoking. Low-grade systemic inflammation induced by smoking may represent the link between these 2 conditions. In this study, we investigated left ventricular dysfunction in patients primarily diagnosed with COPD, and nonreversible airflow limitation in patients primarily diagnosed with CHF. The levels of circulating high-sensitive C-reactive protein (Hs-CRP), pentraxin 3 (PTX3), interleukin-1β (IL-1 β), and soluble type II receptor of IL-1 (sIL-1RII) were also measured as markers of systemic inflammation in these 2 cohorts. Patients aged ≥ 50 years and with ≥ 10 pack years of cigarette smoking who presented with a diagnosis of stable COPD (n=70) or stable CHF (n=124) were recruited. All patients underwent echocardiography, N-terminal pro-hormone of brain natriuretic peptide measurements, and post-bronchodilator spirometry. Plasma levels of Hs-CRP, PTX3, IL-1 β, and sIL-1RII were determined by using a sandwich enzyme-linked immuno-sorbent assay in all patients and in 24 healthy smokers (control subjects). Although we were unable to find a single COPD patient with left ventricular dysfunction, we found nonreversible airflow limitation in 34% of patients with CHF. On the other hand, COPD patients had higher plasma levels of Hs-CRP, IL1 β, and sIL-1RII compared with CHF patients and control subjects (p < 0.05). None of the inflammatory biomarkers was different between CHF patients and control subjects. In conclusion, although the COPD patients had no evidence of CHF, up to one third of patients with CHF had airflow limitation, suggesting that routine spirometry is warranted in patients with CHF, whereas echocardiography is not required in well characterized patients with COPD. Only smokers with COPD seem to have evidence of systemic inflammation.

OBJECTIVE

Nerve growth factor (NGF), a neurotrophin, is induced in lung cells by proinflammatory cytokines, and has a role in bronchial hyperreactivity and lung tissue repair. Ventilation induced lung injury, on the other hand, is known to increase the levels of proinflammatory cytokines in the lungs. We investigated whether, and to what extent, various degrees of lung injury induced by short-term ventilation affect NGF levels in the lung tissue of adolescent rabbits.

METHODS

The rabbits were randomized to different modes of ventilation: (1) CON: normal ventilation for 30 min; (2) NVT: normal ventilation for 6 hr; (3) HFQ: ventilation for 6 hr at double frequency, but normal tidal volume (VT); and (4) HVT: 6 hr ventilation at double VT but normal frequency.

RESULTS

NGF protein was detected in bronchoalveolar lavage fluid (BALF) and lung tissue in all animals. Ventilation for 6 hr significantly increased NGF levels, in both BALF and lung tissue, in the HFQ and HVT groups as compared to control (P < 0.05). The maximum increase in BALF NGF was seen in the HVT group (P = 0.02 vs. CON and NVT groups, and P = 0.05 vs. HFQ). A parallel increase in interleukin 1-beta (IL1-beta) was observed. Expression of the high-affinity NGF-receptor, tropomyosin-related kinase A (TrkA), was also upregulated in these two groups.

CONCLUSIONS

Injurious modes of mechanical ventilation upregulate NGF and its receptor TrkA in rabbit lungs, and IL1-beta may be a mediator for this response. We speculate that this increase in NGF level may translate into the development of bronchial hyperreactivity.

OBJECTIVE

To determine whether interleukin-1 alpha and 1 beta gene polymorphism is associated with rheumatoid arthritis disease activity and bone mineral metabolism, and whether there is any relationship between IL-1 beta and rheumatoid arthritis (RA) motif gene.

METHODS

IL-1 gene polymorphisms were analyzed in 65 RA patients who met American College of Radiology (ACR) criteria and 60 controls. From genomic DNA, 2 polymorphisms in each gene for IL1 alpha-889 and IL-1 beta + 3953 were typed by PCR-RFLP and HLA-DRB1 allele typing was also undertaken by PCR-SSOP. Some clinical and laboratory parameters were collected. The allelic frequencies and carriage rates were compared between RA patients and controls and between patients with active and quiescent disease. Comparison was also made between IL-1 polymorphism and parameters of bone mineral metabolism and between patients with the HLA-DRB1 RA motif plus IL-1 beta 2 and patients without the two alleles. Fisher test and the analysis of variance was used to analyze the data.

RESULTS

There was no significant difference in the frequency and carriage rate of IL-1 alpha polymorphisms between RA patients and the controls. The beta 2/2 genotype of IL-1 beta was more common in female RA patients compared with controls (P = 0.001). A lower carriage rate of IL-1 beta 2 occurred in male RA patients (P = 0.001). A higher carriage rate of IL-1 alpha 2 is associated with a higher ESR (P = 0.008), HAQ score (P = 0.03), and vit-D3 (P < 0.001), but conversely a lower SJC (p = 0.002), a lower RF (P = 0.002) and a lower BMD at the lumbar spine (P = 0.001). A higher frequency of IL-1 alpha 1 is associated with a lower CRP value (P = 0.009). An increased IL-1 beta 2 carriage is associated with active rheumatoid disease as indicated by a higher CRP (P < 0.001), ESR (P < 0.001) and pain score (P = 0.001) and a higher BMD at the lumbar spine (P = 0.007), lower vit-D3 and. Udpd/Crea level The presence of the HLA DRB1 RA motif and IL-1 beta allele 2 at same time did not contribute to disease activity.

CONCLUSIONS

Polymorphisms of the IL-beta gene may affect the RA occurrence. Carriage of IL-1 beta 2 polymorphisms is associated with more active disease in RA and the presence of both the IL-1 alpha 2 and the IL-1 beta 1 allele in RA influences bone resorption.

OBJECTIVE

The pathophysiology of chronic hepatitis C and the mechanisms of resistance to interferon alpha are poorly understood. The aim of this work was to assess the influence of HCV infection and the viral genotype on lymphocyte production of 2',5' oligo-adenylate synthetase activity and monocyte production of TNF alpha and IL1 beta.

METHODS

Mononuclear cells from 50 consecutive patients were studied after 6 months of interferon treatment. Patients with persistent viremia (PCR-positive, elevated ALT, n = 39) were compared with the PCR-negative patients with normal ALT activity (n = 11) of similar age and sex ratio.

RESULTS

Cells from the viremic patients showed lower basal and stimulated 2',5' oligo-adenylate synthetase activity, and a lower in vitro response capacity to human recombinant interferon. In contrast, no difference was observed in basal and stimulated TNF alpha or IL1 beta production between the two groups. In the PCR-positive patients the viral genotype had no significant influence on the response of mononuclear cells to interferon or endotoxin.

CONCLUSIONS

These results show that the presence of HCV in blood is associated with an elective defect in interferon system activation, independently of the viral genotype.

The most studied genetic susceptibility factors involved in gastric carcinoma (GC) risk are polymorphisms in the inflammation-linked genes interleukin 1 (IL1) B and IL1RN. Despite the evidence pointing to the IL1 region, definite functional variants reproducible across populations of different genetic background have not been discovered so far. A high density linkage disequilibrium (LD) map of the IL1 gene cluster was established using HapMap to identify haplotype tagSNPs. Eighty-seven SNPs were genotyped in a Portuguese case-control study (358 cases, 1,485 controls) for the discovery analysis. A replication study, including a subset of those tagSNPs (43), was performed in an independent analysis (EPIC-EurGast) containing individuals from 10 European countries (365 cases, 1284 controls). Single SNP and haplotype block associations were determined for GC overall and anatomopathological subtypes. The most robust association was observed for SNP rs17042407, 16Kb upstream of the IL1A gene. Although several other SNP associations were observed, only the inverse association of rs17042407 allele C with GC of the intestinal type was observed in both studies, retaining significance after multiple testing correction (p = 0.0042) in the combined analysis. The haplotype analysis of the IL1A LD block in the combined dataset revealed the association between a common haplotype carrying the rs17042407 variant and GC, particularly of the intestinal type (p = 3.1 × 10(-5) ) and non cardia localisation (p = 4.6 × 10(-3) ). These results confirm the association of IL1 gene variants with GC and reveal a novel SNP and haplotypes in the IL1A region associated with intestinal type GC in European populations.

Hemorrhagic shock is a common cause of death in emergency rooms. Current animal models of hemorrhage encounter a major problem that the volume and the rate of blood loss cannot be controlled. In addition, the use of anesthesia obscures physiological responses. Our experiments were designed to establish an animal model based on the clinical situation for studying hemorrhagic shock. Hemorrhagic shock was induced by withdrawing blood from a femoral arterial catheter. The blood volume withdrawn was 40% of the total blood volume for group 1 and 30% for group 2 and 3. Group 3 was anesthetized with sodium pentobarbital (25 mg/kg, i.v.) at the beginning of blood withdrawal. Our data showed that the survival rate was 87.5% at 48 h in the conscious group and 0% at 9 h in anesthetic group after hemorrhage. The levels of mean arterial pressure, heart rate, white blood count, TNF-alpha, IL1-beta, CPK, and LDH after blood withdrawal in the anesthetic group were generally lower than those in conscious groups. These results indicated that anesthetics significantly affected the physiology of experimental animals. The conscious, unrestrained and cumulative volume-controlled hemorrhagic shock model was a good experimental model to investigate the physical phenomenon without anesthetic interfernce.

BACKGROUND

Synovial fluid was collected prior to and at 3 to 4 days after ACL reconstruction to investigate the correlation between inflammatory cytokine levels in the acute phase after surgery and physical functional recovery at 3 months postoperatively.

METHODS

For this purpose, 79 patients with ACL reconstruction using semitendinosus tendons were included in the study. Median days from injury to surgery were 80 days (13-291 days). Synovial fluid was obtained just before surgery and at 3 to 4 days after surgery. Physical activity of each patient was evaluated at 3 months postoperatively, and scored from 0 (hard to walk) to 5 (run). Patients able to jog (score 4) or run (score 5) were considered as the "quick recovery" group and others (scores 1-3) as the "delayed recovery" group.

RESULTS

Physical activity recovery scores in the early surgery group (preoperative period less than 60 days; Group I) were significantly better than those in the delayed surgery group (Group II). Among the cytokines tested, TNF-alpha and <em>IL1</em>0 levels in synovial fluid were significantly higher in Group II at 3 to 4 days postoperatively, while levels of these cytokines were quite comparable preoperatively between the groups. Increased <em>IL1</em>-<em>beta</em> expression was noted in the delayed recovery group at 3 to 4 days postoperatively. In addition, levels of IL6, <em>IL1</em>0 and IFN-gamma also tended to increase in patients with delayed recovery.

CONCLUSIONS

Delayed ACL reconstruction increases levels of inflammatory cytokines in synovial fluid after surgery and correlates with a prolonged recovery of short-period physical activity of the patients.

Interleukin-1 beta (IL-1β)-the most potent pro-inflammatory is responsible for a broad spectrum of immune and inflammatory responses, it induces T-cell and B-cell activation and consequently the synthesis of other pro-inflammatory cytokines (such as IFN-γ and TNF). IL-1β induces the formation of blood platelet-leukocyte aggregates (PLAs), which suggests that IL-1β significantly affects the cross-talk between blood platelets and the immune response system, leading to coronary thrombosis. The aim of our study is to investigate the effect of flavonolignans (silybin, silychristin and silydianin) on the IL-1β-induced interaction between platelets and leukocytes, as well as on the expression and the secretion of pro-inflammatory factors. Whole blood samples were pre-incubated with commercially available flavonolignans (silybin, silychristin and silydianin) in a concentration range of 10-100 µM (30 min, 37 °C). Next, samples were activated by IL-1β for 1 h. Blood platelet-leukocyte aggregates were detected by using the double-labeled flow cytometry (CD61/CD45). The level of produced cytokines was estimated via the ELISA immunoenzymatic method. IFN-γ and TNF gene expression was evaluated using Real Time PCR with TaqMan arrays. We observed that in a dose-dependent manner, silybin and silychristin inhibit the IL-1β-induced formation of blood platelet-leukocyte aggregates in whole blood samples, as well as the production of pro-inflammatory cytokines-IL-2, TNF, INF-α, and INF-γ. Additionally, these two flavonolignans abolished the IL-1β-induced expression of mRNA for IFN-γ and TNF. Our current results demonstrate that flavonolignans can be novel compounds used in the prevention of cardiovascular diseases with dual-use action as antiplatelet and anti-inflammatory agents.

OBJECTIVE

We aimed to study the effect of trimetazidine (TMZ) on urethral wound repair.

METHODS

A total of 52 male rats were used; 8 groups were formed: 1-week and 3-week control (C1, C3), sham (S1, S3), oral (OT1, OT3), and intraurethral TMZ (IUT1, IUT3) groups. Serum and urine total antioxidant capacity (TAC), total oxidant capacity (TOC), and 8-hydroxy-deoxy-guanosine (8-OHdG) were studied. Hematoxyline-Eosin was used for the histopathological study. In addition, tumor necrosis factor alpha (TNF- α), interleukin 1α, and β levels were compared across groups by an immunohistochemical method.

RESULTS

There were significant differences between C3 and IUT3, OT3 and IUT3 with respect to serum TAC in 3-week groups (P=0.013; P =0.001). Serum TOC levels were significantly different between C3 and IUT3; S3 and OT3; and OT3 and IUT3 groups (P =0.024; P =0.019; P =0.000, respectively). Serum 8-OHdG levels were significantly different between C3 and OT3 groups (P=0.033). In the immunohistochemical examination, C1 and OT1; C1 and IUT1; and S1, S3, OT1, OT3, IUT1 groups were significantly different with respect to IL-1β staining (P=0.007; P =0.000; P=0.009), while there was a significant difference between C3 and S3 with respect to IL-1β (P =0.000).

CONCLUSIONS

TMZ increased urinary total oxidant level; while increasing serum TAC levels in the long-term. It also reduced serum TAC levels in urethral use and caused an increase in serum TOC levels with minimal effects on DNA injury and repair. No effect was detected on IL1 α and TNF, but partially reduced the effect on IL-1 β levels.

BACKGROUND

Red-fleshed sweet orange juice (ROJ) comes from a new variety of citrus cultivated in Brazil that contains high levels of β-carotene and lycopene, and similar amounts of hesperidin (HSP) and nutrients, equivalently to blond orange juice (BOJ). Such bioactive compounds are associated with chemopreventive actions in several cancer cell lines. The purpose of this study was to examine the cytotoxicity, cell cycle, apoptosis, and cytokine secretion after BOJ, ROJ, and HSP treatment of a novel T acute lymphoblastic leukemia cell line, Loucy.

METHODS

Loucy cells were incubated for 24-h with BOJ, ROJ, and HSP, and the viability was measured using trypan blue. Cell cycling and apoptosis were assessed by propidium iodide (PI) and annexin V-FITC/PI flow cytometry, respectively. Secretion of cytokines IL-1α, IL1-β, IL-2, IL-4, IL-6, IL-10, IL-17A, IFNγ, TNFα, TGFβ, MIPα, and MIPβ was determined by ELISA array.

RESULTS

BOJ and ROJ treatments promoted Loucy cell cytotoxicity. Additionally, BOJ induced cell cycle arrest in the G0/G1 phase, and decreased the cell accumulation in the G2/M. ROJ decreased only the G0/G1 fraction, while HSP did not change the cell cycle. BOJ led to apoptosis in a different fashion of ROJ, while the first treatment induced apoptosis by increase of late apoptosis and primary necrotic fractions, the second increased early and late apoptosis, and primary necrotic fraction compared to positive controls. HSP had no effect on apoptosis. IL-6 and IL-10 were abrogated by all treatments.

CONCLUSIONS

Taking together, these results suggest potential chemopreventive effects of BOJ and ROJ on Loucy cells.

P48 is a 48 kd monocytic differentiation/activation factor previously purified from the conditioned medium of the Reh human pre-B cell leukemia line. It induces differentiation of HL-60 promyelocytic leukemia cells along the monocytic pathway and production of IL1, TNF-alpha and IL6 in human monocytes and monocytic cell lines. Recently our laboratory isolated cDNA clones for P48 from Reh cells and genomic clones from Mycoplasma fermentans DNA and showed that P48 is a M. fermentans gene product. In this paper we report the analysis of P48 expression at the DNA, mRNA and protein levels in different Mycoplasma species. Polymerase Chain Reaction (PCR) analysis of extracted DNA using P48-specific oligonucleotide primers revealed P48 sequences in M. fermentans but not M. hominis, M. iowae, M. genitalium or M. capricolum. Southern analysis of Mycoplasma DNAs revealed hybridizing bands in M. fermentans and M. capricolum under low stringency, but only in M. fermentans under high stringency. Consistent with this, Northern blot studies revealed a single hybridizing transcript in M. fermentans but not in other Mycoplasma species tested. However, Western blot studies with anti-P48 antibodies revealed P48 antigenic material in M. fermentans, as well as M. hominis and M. iowae. These studies demonstrate that the gene for P48 is derived from M. fermentans or a closely related species and is absent in these other species tested. However, the P48 protein exhibits shared antigenic determinants among several Mycoplasma species which presently are of unknown function or significance. P48 is a Mycoplasma -derived immunomodulatory molecule which may be important in Mycoplasma pathophysiology and may be useful in understanding human haematopoietic differentiation and the control of cytokine biosynthesis.

We studied the effect of concentrated surfactant-depleted BALF from 8 normal subjects, 13 patients with sarcoidosis, and 13 patients with AIDS on IL1 beta release by human AM. Adherent target AM were exposed to concentrated BALF in the presence or absence of LPS (1 microgram/ml) for two hours. Control AM were unexposed to BALF. After an additional 24-hour incubation, AM supernatants were collected and measured for IL1 beta by ELISA. No spontaneous IL1 beta release occurred from unstimulated AM. One of the sarcoid-BALF and three of the AIDS-BALF samples induced a small amount of IL1 beta release from unstimulated AM. In LPS-stimulated AM, exposure to normal BALF did not significantly alter IL1 beta release compared to unexposed AM. Exposure to sarcoid-BALF significantly increased the release of IL1 beta, while exposure to AIDS-BALF significantly reduced the IL1 beta level in the AM supernatants. The latter effect was related to the higher mortality induced by AIDS-BALF in AM. These data show that release of IL1 beta from LPS-stimulated AM is modified by a short exposure to a sample of alveolar fluid from patients with lung disease.

UNASSIGNED

To investigate the antimicrobial, immunological and healing effects of Melipona scutellaris honey on infected wounds of rat skin.

METHODS

Twenty four Wistar rats were distributed in four groups (6-each). The uninfected skin wounds of group I rats were treated daily with saline for 7 days. Uninfected wounds (group II) rats were treated with honey. In group III (treated with saline) and group IV (treated with honey) wounds were inoculated with MRSA ATTC43300. The first bacterial culture was performed 24 hours later. In the 7th day new culture was done, and wound biopsies were used for cytokines dosage and histopathology.

RESULTS

In group I and III rats the CFU/g count of S. aureus in wounds was zero. In group II rats the CFU/g counts in the wound tissue were significantly higher than in wounds of group IV rats. The density histopathological parameters and the expression of TNF-α, IL1-β, Il-6 were significantly higher on wounds of group IV then in the other groups.

CONCLUSIONS

Honey of Melipona scutellaris was effective in the management of infected wounds, by significant bacterial growth inhibition, enhancement of cytokine expression, and positively influenced the wound repair.

Snakebites caused by the genus Bothrops are often associated with severe and complex local manifestations such as edema, pain, hemorrhage, and myonecrosis. Conventional treatment minimizes the systemic effects of venom; however, their local action is not neutralized. The purpose of this study was to evaluate the effect of photobiomodulation (PBM) on C2C12 muscle cells exposed to B. jararaca, B. jararacussu, and B. moojeni venoms on events involved in cell death and the release of inflammatory mediators. Cells were exposed to venoms and immediately irradiated with low-level laser (LLL) application in continuous wave at the wavelength of 660 nm, energy density of 4.4 J/cm², power of 10 mW, area of 0.045 cm², and time of 20 s. Cell integrity was analyzed by phase contrast microscope and cell death was performed by flow cytometry. In addition, interleukin IL1-β, IL-6, and IL-10 levels were measured in the supernatant. Our results showed that the application of PBM increases cell viability and decreases cell death by apoptosis and necrosis. Moreover, the release of pro-inflammatory interleukins was also reduced. The data reported here indicate that PBM resulted in cytoprotection on myoblast C2C12 cells after venom exposure. This protection involves the modulation of cell death mechanism and decreased pro-inflammatory cytokine release.

Toll/IL1 receptor (TIR) adaptor proteins continue to be an integral part of Toll-like receptors' (TLR) signalling involved in inflammation. Signalling is likely to be initiated by these TIR adaptors when they are recruited to a TIR-TIR interface formed by TLR dimerization. Among these, myeloid differentiation factor-88 (MyD88), MyD88 adapter-like protein (Mal), TIR domain-containing adaptor protein inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) play pivotal roles at many steps in the signalling events leading to inflammation. The presence of the conserved BB loop residues in the TIR domain of all these important adaptor proteins make them possible targets for inhibition by synthetic compounds. We have designed compounds based on an already known MyD88 TIR dimerization inhibitor, T6167923, which binds well not only to the original target but also to the TIR domains of Mal, TRIF and TRAM. The designed inhibitors are based on modifications of the bromophenyl-sulphonyl-thiophenyl-piperazine-carboxamide series of compounds. We have further suggested modifications in these high-affinity compounds for efficient absorption inside the body. Further, a pharmacophore model highlighting important structural interaction features has been developed. The screened compounds are better in binding to the TIR proteins then the parent compound and hence are good starting points for multi-TIR inhibition.

BACKGROUND

During chemotherapy, the correlation between insomnia and fatigue, anxiety, pain, depressed mood, and cognitive disorders makes these subjective complaints a 'symptom cluster' with common biological mechanisms. The theory of cerebral inflammation following the production of pro-inflammatory cytokines (high level of interleukin 1-β [IL1-β], IL6 and tumour necrosis factor-alpha) is currently the most generally accepted. Understanding these mechanisms should allow us to propose a chemoprotective homeopathic treatment of the nervous system.

METHODS

By retaining the inflammatory aetiology, we combined the rubrics 'Inflammation of the brain', 'Inflammation of the meninges', 'Inflammation of the nerves' with the symptom cluster: insomnia, fatigue, depressive state and memory disorders.

RESULTS

After repertorisation, we propose the following homeopathic protocol: Belladonna 15c, Phosphorus 15c, Cerebral cortex 4c and Nerves 4c, two pills of each medicine to be sucked together before breakfast, lunch and dinner, on each day of chemotherapy and for the following 2 days.

CONCLUSIONS

This selected protocol, derived from a physiopathological knowledge of the symptoms, seems to be well suited to the prevention and treatment of post-chemotherapeutic cerebral inflammation. It is essential to start the homeopathic treatment before the chemotherapy session to anticipate the emergence of the 'chemo-brain' side effects. This proposed prevention protocol must be confirmed and quantified by randomised studies.

The role of chitinases from the latex of medicinal shrub Calotropis procera on viability of tumor cell lines and inflammation was investigated. Soluble latex proteins were fractionated in a CM Sepharose Fast-Flow Column and the major peak (LPp1) subjected to ion exchange chromatography using a Mono-Q column coupled to an FPLC system. In a first series of experiments, immortalized macrophages were cultured with LPp1 for 24 h. Then, cytotoxicity of chitinase isoforms (LPp1-P1 to P6) was evaluated against HCT-116 (colon carcinoma), OVCAR-8 (ovarian carcinoma), and SF-295 (glioblastoma) tumor cell lines in 96-well plates. Cytotoxic chitinases had its anti-inflammatory potential assessed through the mouse peritonitis model. We have shown that LPp1 was not toxic to macrophages at dosages lower than 125 μg/mL but induced high messenger RNA expression of IL-6, IL1-β, TNF-α, and iNOs. On the other hand, chitinase isoform LPp1-P4 retained all LPp1 cytotoxic activities against the tumor cell lines with IC50 ranging from 1.2 to 2.9 μg/mL. The intravenous administration of LPp1-P4 to mouse impaired neutrophil infiltration into the peritoneal cavity induced by carrageenan. Although the contents of pro-inflammatory cytokines IL-6, TNF-α, and IL1-β were high in the bloodstreams, such effect was reverted by administration of iNOs inhibitors NG-nitro-L-arginine methyl ester and aminoguanidine. We conclude that chitinase isoform LPp1-P4 was highly cytotoxic to tumor cell lines and capable to reduce inflammation by an iNOs-derived NO mechanism.

Signaling mediated by cytokines and chemokines is involved in glaucoma-associated neuroinflammation and in the damage of retinal ganglion cells (RGCs). Using multiplexed immunoassay and immunohistochemical techniques in a glaucoma mouse model at different time points after ocular hypertension (OHT), we analyzed (i) the expression of pro-inflammatory cytokines, anti-inflammatory cytokines, BDNF, VEGF, and fractalkine; and (ii) the number of Brn3a+ RGCs. In OHT eyes, there was an upregulation of (i) IFN-γ at days 3, 5, and 15; (ii) IL-4 at days 1, 3, 5, and 7 and IL-10 at days 3 and 5 (coinciding with downregulation of IL1-β at days 1, 5, and 7); (iii) IL-6 at days 1, 3, and 5; (iv) fractalkine and VEGF at day 1; and (v) BDNF at days 1, 3, 7, and 15. In contralateral eyes, there were (i) an upregulation of IL-1β at days 1 and 3 and a downregulation at day 7, coinciding with the downregulation of IL4 at days 3 and 5 and the upregulation at day 7; (ii) an upregulation of IL-6 at days 1, 5, and 7 and a downregulation at 15 days; (iii) an upregulation of IL-10 at days 3 and 7; and (iv) an upregulation of IL-17 at day 15. In OHT eyes, there was a reduction in the Brn3a+ RGCs number at days 3, 5, 7, and 15. OHT changes cytokine levels in both OHT and contralateral eyes at different time points after OHT induction, confirming the immune system involvement in glaucomatous neurodegeneration.

Keywords: BDNF; Brn3a; Iba-1; VEGF; cytokines; fractalkine; glaucoma; microglia; ocular hypertension; retinal ganglion cells (RGCs).

Study question: Can antioxidant treatment with N-acetylcysteine (NAC) protect ovarian follicles from ischemia-reperfusion injury in xenotransplanted human ovarian tissue?

Summary answer: Daily administration of NAC for 7-12 days post-transplantation reduced ischemia-reperfusion injury and increased follicle survival in human ovarian xenografts by upregulating the antioxidant defense system and exerting anti-inflammatory and antiapoptotic effects.

What is known already: Freezing of human ovarian tissue is performed with high follicular survival rates but up to 70% of follicles appear to be lost due to hypoxia and ischemia-reperfusion injury during ovarian tissue transplantation (OTT). NAC has been demonstrated to possess antioxidant and antiapoptotic properties, and studies in rodents have shown that intraperitoneal administration of NAC reduces ischemia-reperfusion injury and increases follicle survival in autotransplanted murine ovaries.

Study design, size, duration: Pieces of frozen-thawed human ovarian tissue from 28 women aged 23-36 years were transplanted to immunodeficient mice in short- and long-term xenograft studies or cultured in vitro. Three short-term xenograft studies (1-week duration) were performed, in which saline or 150 mg/kg NAC was administered for 7 days post-transplantation (n = 12 patients per group). Two long-term xenograft studies (4 weeks of duration) were performed. In one of these studies, saline or 150 mg/kg NAC was administered for 12 days (n = 12 patients per group), while in the other study 50, 150 or 300 mg/kg NAC was administered for 7 days (n = 8 patients per group). In addition, human ovarian tissue (n = 12 pieces from three patients per group) was cultured with increasing concentrations of NAC (0, 5, 25 and 75 mM) for 4 days in vitro.

Participants/materials, setting, methods: Donated ovarian tissue was obtained from women who had undergone ovarian tissue cryopreservation for fertility preservation at the University Hospital of Copenhagen. Cortical tissue pieces (5 × 5 × 1 mm) were transplanted subcutaneously to immunodeficient mice and NAC or saline was injected intraperitoneally. Grafts were retrieved after 1 or 4 weeks and follicle density was assessed. Gene expression analysis of antioxidant defense markers (superoxide dismutase; Sod1/SOD1, heme oxygenase-1; Hmox1/HMOX1, catalase; Cat/CAT), proinflammatory cytokines (tumor necrosis factor-alpha; Tnf-α, interleukin-1-beta; Il1-β, interleukin 6; Il6), apoptotic factors (B-cell lymphoma 2; Bcl2/BCL2, Bcl-2-associated X protein; Bax/BAX) and angiogenic factors (vascular endothelial growth factor A; Vegfa/VEGFA, angiopoietin-like 4; Angptl4/ANGPTL4) was performed in 1-week-old human ovarian xenografts and in cultured human ovarian tissue. Grafts retrieved after 4 weeks were histologically processed and analyzed for vascularization by CD31 immunohistochemical staining, fibrosis by Masson's Trichrome staining and apoptosis by immunofluorescence using cleaved caspase-3.

Main results and the role of chance: After 1-week grafting, the relative expression of Sod1, Hmox1 and Cat was significantly higher in the group receiving 150 mg/kg NAC (NAC150-treated group) compared to controls (P = 0.04, P = 0.03, and P = 0.01, respectively), whereas the expression levels of Tnf-α, Il1-β and Il6 were reduced. The Bax/Bcl2 ratio was also significantly reduced in the NAC150-treated group (P < 0.005). In vitro, the relative gene expression of SOD1, HMOX1 and CAT increased significantly in the human ovarian tissue with increasing concentrations of NAC (P < 0.001 for all genes). However, the expression of VEGFA and ANGPTL4 as well as the BAX/BCL2 ratio decreased significantly with increasing concentrations of NAC (P < 0.02, P < 0.001 and P < 0.001, respectively). After 4-week grafting, fibrosis measured by collagen content was similar in the NAC150-treated group compared to controls (control: 56.6% ± 2.2; NAC150: 57.6% ± 1.8), whereas a statistically significant reduction in the CD31-positive vessel area was found (control: 0.69% ± 0.08; NAC150: 0.51% ± 0.07; P < 0.02). Furthermore, a reduced immunoreactivity of cleaved caspase-3 was observed in follicles of the NAC150-treated xenografts compared to controls. Follicle density (follicles/mm3, mean ± SD) was higher in the NAC150-treated group compared to the control group in the 1-week xenografts (control: 19.5 ± 26.3; NAC150: 34.2 ± 53.5) and 4-week xenografts (control: 9.3 ± 11.0; NAC150: 14.4 ± 15.0). Overall, a 2-fold increase in follicle density was observed in the NAC150-group after 1-week grafting where fold changes in follicle density were calculated in relation to grafts from the same patient. Around a 5-fold increase in follicle density was observed in the NAC150 and NAC300 groups after 4-week grafting.

Large scale data: N/A.

Limitations, reasons for caution: Follicle density in the human ovarian cortex is highly heterogeneous and can vary 100-fold between cortex pieces from the same woman. A high variability in follicle density within and between treatment groups and patients was found in the current study. Thus, solid conclusions cannot be made. While intraperitoneal injections of NAC appeared to reduce ischemia-reperfusion injury in human ovarian xenografts, different administration routes should be investigated in order to optimize NAC for potential clinical use.

Wider implications of the findings: This is the first study to demonstrate the antioxidant, anti-inflammatory and antiapoptotic properties of NAC in xenotransplanted human ovarian tissue. Therefore, NAC appears to be a promising candidate for protecting ovarian follicles from ischemia-reperfusion injury. This provides the initial steps toward clinical application of NAC, which could potentially reduce the loss of ovarian follicles following OTT.

Study funding/competing interest(s): We are grateful to the Danish Childhood Cancer Foundation, Hørslev Foundation, Aase and Einar Danielsen's Foundation (grant number: 10-001999), Dagmar Marshalls Foundation, Else and Mogens Wedell-Wedellsborgs Foundation, Knud and Edith Eriksens Mindefond, and Fabrikant Einar Willumsens Mindelegat for funding this study. None of the authors have any competing interests to declare.

Keywords: N-acetylcysteine; antioxidant defense; follicle survival; ischemia-reperfusion injury; ovarian tissue transplantation.

The aim of this paper was to screen out relevant genes of geniposide-induced hepatotoxicity based on genomics,in order to provide a scientific basis for the non-clinical evaluation of drugs containing Gardeniae Fructus and geniposide. Fifty-five SD rats were randomly divided into normal control group,24 h group and 72 h group. The changes of appearance,behavior and weight of rats were observed after administration by gavage for 3 days. The activities of ALT and AST were detected. Molecular mechanism of geniposideinduced hepatotoxicity was investigated by Affymetrix miRNA 4. 0 and Affymetrix Rat Gene 2. 0 to examine the gene expression levels in Sprague-Dawley rat livers at 24 h and 72 h after administration of overdose-geniposide( 300 mg·kg-1 daily),and then verified by Realtime quantitative PCR. Compared with the normal control group,the activities of ALT and AST were markedly increased. In addition,experimental results indicated that 324 genes were differentially expressed,among which 259 were up-regulated and 65 down-regulated.Nine candidate genes were verified by qRT-PCR,including Bcl2,Il1 b,Tpm3,MMP2,Col1α1,Ifit1,Aldob,Nr0 bb,Nr0 bbe correlated with geniposide-induced hepatotoxicity. This study provides an important clue for mechanism of geniposide-induced hepatotoxicity.

We have examined the expression of a panel of cytokines in thymic epithelial cells and CD4-CD8- (DN) thymocytes following cell to cell lymphostromal interaction, in an experimental model which enhances in vitro thymocyte maturation. Since retinoic acid (RA) has been previously shown to be an inhibitor of thymocyte maturation process in this model, we wanted to analyse cytokine expression in DN thymocytes and thymic epithelial cells following the RA-induced impairment of in vitro thymocyte maturation. Cell to cell lymphostromal interaction results in increased IL2 and decreased IL7 expression in thymocytes while the expression of IL1 beta and IL7 increased and decreased, respectively, in thymic epithelial cells. Addition of RA to lympho-stromal cell co-culture results in the enhancement of IL4 and IL7 expression in thymocytes while in thymic epithelial cells IL1 alpha decreased and IL6 and IL7 increased. These data indicate that discrete patterns of cytokine expression are present in thymocyte precursors and in thymic epithelial cells during in vitro T-cell development. They furthermore suggest that specific cytokine modulation might contribute to the RA-induced impairment of thymocyte differentiation.

Lymphocyte stimulation and proliferation play a pivotal role in the immune response to soluble as well as to cellular, bacterial, and viral antigens. In this study, peripheral blood mononuclear cells (PBMC), mainly composed of lymphocytes, were separated by Ficoll-Hypaque density gradient centrifugation from 50-ml jugular vein blood samples drawn from six captive and five wild-caught brown bears (Ursus arctos) (eight Apennine brown bears from the Italian population; three of undetermined origin). Stimulation of cultured bear PBMC with the two classical T lymphocyte mitogens phytohemagglutinin (PHA) and Concanavalin A (ConA) was followed by a significantly greater proliferative response than that shown by human PBMC (n = 11) (PHA: T = 4.03, d.f. = 20, P = 0.001; ConA: T = 4.25, d.f. = 20, P < 0.0005; Student's t-test, oneway ANOVA). As in humans, the PBMC proliferative response in bears was markedly >> 50%) inhibited by addition of transforming growth factor beta (TGF beta) human recombinant cytokine to the culture. Further fractionation provided a cell preparation extremely rich in peripheral blood lymphocytes (PBL) (mean +/- SD = 96.1 +/- 1.7%). Addition of interleukin 1 (IL1) or interleukin 2 (IL2) human recombinant cytokines to cultured PBL stimulated with a suboptimal concentration of mitogens resulted in a ninefold increase in the lymphocyte proliferative response. Dexamethasone (DEX, a synthetic analog of hydrocortisone) inhibited the bear PBMC proliferative response by 22.2 +/- 4.3% (mean +/- SD), compared with 46.2 +/- 6.9% and 91.8 +/- 8.1% (mean +/- SD) in humans and mice (n = 11) (Mus domesticus), respectively. Inhibition of the brown bear and human PBMC responses was markedly >> 60%) reduced by the addition of IL2. The finding that IL1 and IL2 augment and that DEX inhibits bear lymphocyte proliferative response suggests that these cytokines can be used to increase the immune response in vaccinations, and that DEX may hamper several immunologically mediated diseases.

Protein kinase C (PKC) is a family of enzymes detected in a diverse range of cell types where they regulate various cellular functions such as proliferation, differentiation, cytoskeletal remodelling, cytokine production, and receptor-mediated signal transduction. In this study we have analyzed the expression of 11 PKC isoforms (-alpha, -beta(I), -beta(II), -gamma, -delta, -eta, -theta, -epsilon, -zeta, -iota/lambda, and -micro) in osteoblasts from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) in comparison with osteoblasts from post-traumatic (PT) patients. By Western blotting analysis, nine isoforms, -alpha, -beta(I), -beta(II), -delta, -theta, - epsilon, -zeta, - iota/lambda, and -micro, were detected in osteoblasts. In RA and OA patients, PKC -theta and -micro were greater expressed whereas PKC-epsilon and -zeta decreased when compared with normal cells. The subcellular distribution and quantitative differences were confirmed by immuno-electron microscopy. Furthermore, we demonstrated that treatment with the proinflammatory cytokines, IL-1beta and TNF-alpha, significantly decreased PKC-zeta expression in PT osteoblasts. This suggests that proinflammatory cytokines can modulate the expression of this PKC isoform in osteoblasts in a way which is similar to changes detected in arthritic patients.

OBJECTIVE

Intestinal dysmotility and immune activation are likely involved in the pathogenesis of small intestinal bacteria overgrowth (SIBO) in irritable bowel syndrome (IBS). We aimed at investigating the role of interstitial cells of Cajal (ICC) and intestinal inflammation in the development of SIBO using a post-infectious IBS (PI-IBS) mouse model.

METHODS

NIH mice were randomly infected with Trichinella spiralis. Visceral sensitivity and stool pattern were assessed at 8-weeks post-infection (PI). Intestinal bacteria counts from jejunum and ileum were measured by quantitative real-time PCR to evaluate the presence of SIBO. ICC density, intraepithelial lymphocytes (IELs) counts, and intestinal cytokine levels (IL1-β, IL-6, toll-like receptor-4 (TLR-4), IL-10) in the ileum were examined.

RESULTS

PI-IBS mice demonstrated increased visceral sensitivity compared with the control group. One-third of the PI-IBS mice developed SIBO (SIBO+/PI-IBS) and was more likely to have abnormal stool form compared with SIBO negative PI-IBS (SIBO-/PI-IBS) mice but without difference in visceral sensitivity. SIBO+/PI-IBS mice had decreased ICC density and increased IELs counts in the ileum compared with SIBO-/PI-IBS mice. No difference in inflammatory cytokine expression levels were detected among the groups except for increased TLR-4 in PI-IBS mice compared with the control group.

CONCLUSIONS

Development of SIBO in PI-IBS mice was associated with reduced ICC density and increased IELs counts in the ileum. Our findings support the role of intestinal dysmotility and inflammation in the pathogenesis of SIBO in IBS and may provide potential therapeutic targets.