Mechanism of immune dysfunction in sepsis: inducible nitric oxide-meditated alterations in p38 MAPK activation

BACKGROUND: After the onset of sepsis, there is a marked dysfunction in cell-mediated immunity that contributes to the morbidity and mortality seen in this condition. Although both nitric oxide (NO) from inducible NO synthase (iNOS) and the activation of p38 mitogen-activated protein kinase (p38 MAPK) appear to contribute to this immune dysfunction, the extent to which NO regulates p38 MAPK activity in sepsis remains unknown. METHODS: To examine this, we induced sepsis by cecal ligation and puncture (CLP) in iNOS knockout (iNOS -/-) or C57BL/6 control mice. Twenty-four hours after CLP or sham operation, splenic T cells and macrophages were isolated and then stimulated with monoclonal antibody against the T-cell marker CD3 (anti-CD3) or lipopolysaccharide. At 4 or 24 hours after stimulation, cytokine release was determined by enzyme-linked immunosorbent assay, and p38 MAPK phosphorylation (activation) was determined by immunoblotting with antibody specific to phosphorylated p38 MAPK. RESULTS: Splenic T-cell p38 MAPK activation and interleukin (IL)-10 release was increased by CLP, whereas Th1 cytokine (IL-2, interferon-gamma) release was depressed. iNOS gene deficiency inhibited p38 MAPK activation in splenic T cells taken from septic mice, and also suppressed IL-10 release in both sham and septic mice. Interestingly, although deficiency of iNOS restored IL-2 release after CLP, both sham and CLP T cells remained depressed in their ability to release interferon-gamma. Septic insult markedly suppressed C57BL/6 splenic macrophage release of proinflammatory agents tumor necrosis factor, IL-12, and IL-1, while augmenting the release of IL-10. However, although deficiency of iNOS concomitantly restored the ability to produce tumor necrosis factor while suppressing the rise in IL-10 release and p38 MAPK activation, it only partially restored IL-1 release and had no effect on IL-12 production seen after CLP. CONCLUSION: These data suggest that NO release from iNOS regulates aspects of sepsis-induced immune dysfunction by the activation of p38 MAPK.     

The incidence of prostate cancer in a screening population with a serum prostate specific antigen between 2.5 and 4.0 ng/ml: relation to biopsy strategy

PURPOSE: It has recently been suggested that the diagnostic threshold for the prostate specific antigen (PSA) assay be lowered to enhance prostate cancer detection. A 22% incidence of prostate cancer has been reported in men with PSA between 2.5 and 4.0 ng/ml. We designed a study to confirm this observation. MATERIALS AND METHODS: Men who participated in our free early detection program and who had serum PSA between 2.5 and 4.0 ng/ml were asked to undergo prostate biopsy. Of 268 eligible men 151 (56%) agreed to participate in this free trial. All men underwent biopsy using an 11-core multisite directed biopsy scheme. All biopsy cores were color coded for location specificity and examined by 1 pathologist. RESULTS: Cancer was identified in 24.5% (37 of 151) of the men biopsied. The median age of men with cancer was 62 years (range 43 to 74). Conventional systematic sextant biopsies, which accounted for 6 of the 11 cores, detected 73.0% (27 of 37) of the cancers and the alternate site biopsies identified the remaining 10. Gleason score was 6 in 25 men, 3 + 4 in 5, 4 + 3 in 4 and 8 or greater in 3 (median Gleason score 6). There were 14 men who had 1 core positive for cancer, 9 had 2 and 14 had more than 2 (median number of positive cores 2). Of the 14 men with 1 positive core 11 had a less than 3 mm focus of cancer and 8 had only a positive alternate site biopsy. There were 11 cases of abnormal results on digital rectal examination, 5 of which were cancer, and 31 cases of abnormal results on ultrasonography, 13 of which were cancer. Median biological variability in PSA was +/-15% (range 0.4% to 440.0%). CONCLUSIONS: We found a significant incidence of cancer (24.5%, 37 of 51) in men with serum PSA between 2.5 and 4.0 ng/ml. In our study 67.6% of the detected cancers were significant based on the biopsy data. If the PSA threshold is lowered the conventional systematic sextant technique may be preferable to an extended strategy.     

The balance between pro‐ and anti‐inflammatory cytokines is crucial in human allergic contact dermatitis pathogenesis: the role of IL‐1 family members

The interleukin (IL)‐1 family includes 11 members that are important in inflammatory processes. It includes various agonists and two antagonists, IL‐1Ra and IL‐36Ra. Our aim was to investigate whether the IL‐1 family is involved in allergic contact dermatitis (ACD). The expression of IL‐1 family members was evaluated by PCR and immunohistochemistry in the positive patch test reaction site (involved skin) and in the uninvolved skin of ACD patients. We also examined these cytokines in an ex vivo model of ACD. The antagonistic activity of IL‐36Ra was evaluated by injecting recombinant IL‐36Ra in uninvolved skin biopsies of ACD patients. IL‐1Ra and IL‐36Ra expression was quantified in mononuclear cells of nickel‐sensitized patients challenged in vitro with nickel. IL‐33 involvement in ACD was investigated by intra‐dermal injection of anti‐IL‐33 in the uninvolved skin of patients ex vivo. Results showed that IL‐1β, IL‐1Ra, IL‐36α, IL‐36β, IL‐36γ and IL‐33 expression, but not IL‐36Ra expression, was enhanced in ACD‐involved skin. Immunohistochemical analysis and ex vivo skin cultures confirmed these results. Injection of anti‐IL‐33 in ACD‐uninvolved skin inhibited IL‐8 expression, whereas IL‐36Ra inhibited IL‐36α, IL‐36β, IL‐36γ and IL‐8 expression. Nickel induced IL‐1Ra expression in lymphocytes of nickel‐sensitized patients. Hence, various IL‐1 agonists and antagonists may be involved in ACD pathogenesis.     

Maximum tolerable dose for avoidance of cataract after repeated exposure to ultraviolet radiation in rats

The purpose of the present study was to determine the impact of inter-exposure interval between repeated equivalent exposures of ultraviolet radiation (UVR) on threshold accumulated dose for cataract development. Female Sprague-Dawley rats were randomly divided into 5 inter-exposure interval groups with 20 rats in each group. The inter-exposure intervals were 6 h, 1, 3, 9 and 30 days respectively. Each inter-exposure interval group was divided into 5 dose-subgroups. Only one eye of each rat was exposed to ultraviolet radiation (lambdamax=300 nm). The total dose incident on the cornea, in each subgroup varied between 0 approximately 10 kJ/m2. One week after the second exposure, the rats were sacrificed and both lenses were extracted. The intensity of forward light scattering was measured and macroscopic morphology was documented. Maximum tolerable dose (MTD) for each inter-exposure interval was estimated based on the experimentally determined dose-response function. The difference of intensity of light scattering between exposed and contralateral non-exposed lens decreased as a function of inter-exposure interval between the two equivalent exposures. The accumulated MTD2.3:16 was 5.3, 5.1, 5.4, 5.8, and 6.0 kJ/m2 UVR-B for the 6 h, 1, 3, 9 and 30 day inter-exposure interval between the two exposures, respectively. The shorter the inter-exposure interval between two subsequent exposures, the more damage. The time constant for repair of lens damage after in vivo exposure to close to threshold dose was estimated to be eight days and the fraction of repairable damage to be 20%. The accumulated threshold dose for damage after two repeated equivalent exposures to UVR-B increases as a function of inter-exposure interval up to at least 30 days inter-exposure interval.     

Corneal activation of prothrombin to form thrombin, independent of vascular injury

PURPOSE: Two major functions of thrombin observed in the cornea are activation of thrombin-sensitive, proteinase-activated receptors and cleavage of fibrinogen to fibrin. The purpose of this study was to determine whether the normal human cornea itself is competent to convert prothrombin to thrombin and synthesizes the mRNA for the proteins required. METHODS: Human corneas were processed for immunolocalization studies or separated into epithelial, stromal, and endothelial layers for proteins and RNA isolation. The protein extracts were used for Western blots, prothrombin time, and activated partial thromboplastin time assays and fibrinopeptide A generation tests. RNA was used for RT-PCR. Apoptosis of cultured human corneal cells was induced with sodium nitroprusside or camptothecin and activation of prothrombin tested. RESULTS: Prothrombin and its mRNA were present in all three layers of human donor cornea. It was found to be associated with the cells and the extracellular matrix at similar levels across the cornea. With corneal stromal extracts, activation of either the intrinsic or extrinsic coagulation pathways resulted in thrombin activation and fibrin formation with fibrinopeptide A release. Detection of key components of the coagulation cascades confirmed noninjured human corneas contain factors required for prothrombin activation. In addition, mRNAs for representative factors and inhibitors were detected by RT-PCR and confirmed by sequencing. Apoptotic corneal stromal cells provide a surface for prothrombin activation. CONCLUSIONS: These studies suggest that the normal avascular human cornea contains and synthesizes the components required for thrombin generation and that this process does not depend on a breech in the limbal vascular endothelium.     

Verruciform xanthoma of the vulva: case report.

A rare case of verruciform xanthoma of the vulva is reported. Diagnosis was made possible by histopathological examination and immunohistochemical staining. Verruciform xanthomas generally occur in the oral cavity. To the best of our knowledge, this is the third reported case of the tumour located on the genital mucosa. Immunohistochemical study supported the histiocytic origin of the lesion. Clinically, verruciform xanthomas may mimic other verrucous lesions of the vulva, such as seborrhoeic keratosis, verruca simplex, condyloma acuminatum, verrucous carcinoma, or erythropasia of Queyrat, or conditions such as histiocytosis, cutaneous lipidosis, or granular cell myoblastoma. The characteristics that differentiate those conditions from verruciform xanthoma, which can be seen only on histology, are given.     

Methimazole Slows Hepatocyte Streaming in Rats

Hepatocytes are hypothesized to continuallystream from the portal tract to the terminal hepaticvein. By this model, when a cell divides, one of itsprogeny replaces the dividing ancestor and the other is displaced into a more remote location. Thepresent experiment aims to demonstrate thathypothyroidism affects liver cell turnover. Thirty maleadult rats were divided into two groups. One receivedmethimazole for two weeks and the other served as control.Each rat was injected intraperitoneally with 18.5 KBq[³H]thymidine/g body weight. Rats were killedafter 1 hr and two and four weeks. Autoradiography was done. The distance of the labeled cells fromthe portal tract was measured. The mean TSH levels ofthe methimazole-treated group and controls were 1.45 and0.25 mM/liter, respectively (P < 0.01). Hepatocyte streaming was lower in hypothyroid (1.8m/day) than in untreated rats (2.5 m/day) (P< 0.01). The respective labeling indices 1 hr afterlabeling were 0.9% and 1.24% (P < 0.05). We concludethat hypothyroidism diminishes hepatocyte and littoral cellturnover and slows down their streaming.     

Angiosarcoma of the small intestine: an immunoperoxidase study

A case of multifocal angiosarcoma of the small intestine is reported. These tumors often contain areas of solid sheets of cells and may be confused with poorly differentiated carcinoma or other sarcomas, as was the tumor in the reported case. Factor VIII-related antigen was demonstrated in biopsy samples by the peroxidase antiperoxidase method, showing the tumor to be an angiosarcoma. We conclude that the demonstration of factor VIII-related antigen in the neoplastic cells is an important aid in the diagnosis of angiosarcoma.