The importin beta superfamily member RanBP17 exhibits a role in cell proliferation and is associated with improved survival of patients with HPV+ HNSCC.

BACKGROUND: More than twenty years after its discovery, the role of the importin beta superfamily member Ran GTP-binding protein (RanBP) 17 is still ill defined. Previously, we observed notable RanBP17 RNA expression levels in head and neck squamous cell carcinoma (HNSCC) cell lines with disruptive TP53 mutations. METHODS: We deployed HNSCC cell lines as well as cell lines from other tumor entities such as HCT116, MDA-MB-231 and H460, which were derived from colon, breast and lung cancers respectively. RNAi was used to evaluate the effect of RanBP17 on cell proliferation. FACS analysis was used for cell sorting according to their respective cell cycle phase and for BrdU assays. Immunocytochemistry was deployed for colocalization studies of RanBP17 with Nucleolin and SC35 (nuclear speckles) domains. TCGA analysis was performed for prognostic assessment and correlation analysis of RanBP17 in HNSCC patients. RESULTS: RNAi knockdown of RanBP17, significantly reduced cell proliferation in HNSCC cell lines. This effect was also seen in the HNSCC unrelated cell lines HCT116 and MDA-MB-231. Similarly, inhibiting cell proliferation with cisplatin reduced RanBP17 in keratinocytes but lead to induction in tumor cell lines. A similar observation was made in tumor cell lines after treatment with the EGFR kinase inhibitor AG1478. In addition to previous reports, showing colocalization of RanBP17 with SC35 domains, we observed colocalization of RanBP17 to nuclear bodies that are distinct from nucleoli and SC35 domains. Interestingly, for HPV positive but not HPV negative HNSCC, TCGA data base analysis revealed a strong positive correlation of RanBP17 RNA with patient survival and CDKN2A. CONCLUSIONS: Our data point to a role of RanBP17 in proliferation of HNSCC and other epithelial cells. Furthermore, RanBP17 could potentially serve as a novel prognostic marker for HNSCC patients. However, we noted a major discrepancy between RanBP17 RNA and protein expression levels with the used antibodies. These observations could be explained by the presence of additional RanBP17 splice isoforms and more so of non-coding circular RanBP17 RNA species. These aspects need to be addressed in more detail by future studies.

Therapeutic Treatment of Superoxide Dismutase 1 (G93A) Amyotrophic Lateral Sclerosis Model Mice with Medical Ozone Decelerates Trigeminal Motor Neuron Degeneration, Attenuates Microglial Proliferation, and Preserves Monocyte Levels in Mesenteric Lymph Nodes.

Amyotrophic lateral sclerosis (ALS) is an incurable and lethal neurodegenerative disease in which progressive motor neuron loss and associated inflammation represent major pathology hallmarks. Both the prevention of neuronal loss and neuro-destructive inflammation are still unmet challenges. Medical ozone, an ozonized oxygen mixture (O3/O2), has been shown to elicit profound immunomodulatory effects in peripheral organs, and beneficial effects in the aging brain. We investigated, in a preclinical drug testing approach, the therapeutic potential of a five-day O3/O2i.p. treatment regime at the beginning of the symptomatic disease phase in the superoxide dismutase (SOD1G93A) ALS mouse model. Clinical assessment of SOD1G93A mice revealed no benefit of medical ozone treatment over sham with respect to gross body weight, motor performance, disease duration, or survival. In the brainstem of end stage SOD1G93A mice, however, neurodegeneration was found decelerated, and SOD1-related vacuolization was reduced in the motor trigeminal nucleus in the O3/O2 treatment group when compared to sham-treated mice. In addition, microglia proliferation was less pronounced in the brainstem, while the hypertrophy of astroglia remained largely unaffected. Finally, monocyte numbers were reduced in the blood, spleen, and mesenteric lymph nodes at postnatal day 60 in SOD1G93A mice. A further decrease in monocyte numbers seen in mesenteric lymph nodes from sham-treated SOD1G93A mice at an advanced disease stage, however, was prevented by medical ozone treatment. Collectively, our study revealed a select neuroprotective and possibly anti-inflammatory capacity for medical ozone when applied as a therapeutic agent in SOD1G93A ALS mice.

Evaluation of digital image analysis as a supportive tool for the stratification of head and neck vascular anomalies.

BACKGROUND: The histological differentiation of individual types of vascular anomalies (VA), such as lymphatic malformations (LM), hemangioma (Hem), paraganglioma (PG), venous malformations (VeM), arteriovenous malformations (AVM), pyogenic granulomas (GP), and (not otherwise classified) vascular malformations (VM n.o.c.) is frequently difficult due to the heterogeneity of these anomalies. The aim of the study was to evaluate digital image analysis as a method for VA stratification METHODS: A total of 40 VA tissues were examined immunohistologically using a selection of five vascular endothelial-associated markers (CD31, CD34, CLDN5, PDPN, VIM). The staining results were documented microscopically followed by digital image analyses based quantification of the candidate-marker-proteins using the open source program ImageJ/Fiji. RESULTS: Differences in the expression patterns of the candidate proteins could be detected particularly when deploying the quotient of the quantified immunohistochemical signal values. Deploying signal marker quotients, LM could be fully distinguished from all other tested tissue types. GP achieved stratification from LM, Hem, VM, PG and AVM tissues, whereas Hem, PG, VM and AVM exhibited significantly different signal marker quotients compared with LM and GP tissues. CONCLUSION: Although stratification of different VA from each other was only achieved in part with the markers used, the results of this study strongly support the usefulness of digital image analysis for the stratification of VA. Against the background of upcoming new diagnostic techniques involving artificial intelligence and deep (machine) learning, our data serve as a paradigm of how digital evaluation methods can be deployed to support diagnostic decision making in the field of VAs.

Indication for differential sorting of the rat v-SNARE splice isoforms VAMP-1a and -1b.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are essential constituents of the intracellular trafficking machinery. The variable C-terminus in the 2 rat VAMP-1 splice isoforms VAMP-1a and -1b potentially acts as a sorting signal, because similar changes at the C-terminal end of a human VAMP-1 splice isoform resulted in its sorting to mitochondria. To evaluate the differences in the subcellular localization of these two v-SNARE proteins, VAMP-1a and -1b proteins tagged with green fluorescent protein (GFP) and red fluorescent protein (RFP) were expressed in HeLa, COS-7, and MDCK cells and evaluated by conventional confocal as well as total internal reflection fluorescence microscopy. Regions consistent with the endoplasmic reticulum and Golgi apparatus demonstrated a major overlap of both signals. In the periphery, vesicular structures were observed that mainly expressed one of the 2 isoforms. Within our experimental settings, we could not observe sorting of any of the 2 isoforms to mitochondria or peroxisomes, whereas both isoforms were found expressed in a minor subset of singular vesicles, which sporadically appeared to co-localize with the exocyst marker EXOC3/Sec6. Because vesicular structures were seen that expressed only one of the two splice variants, it is possible that VAMP-1a and VAMP-1b are sorted to distinct cellular compartments that require further characterization.

Epidermal growth factor-induced modulation of cytokeratin expression levels influences the morphological phenotype of head and neck squamous cell carcinoma cells.

The migratory ability of tumor cells requires cytoskeletal rearrangement processes. Epidermal growth factor receptor (EGFR)-signaling tightly correlates with tumor progression in head and neck squamous cell carcinomas (HNSCCs), and has previously been implicated in the regulation of cytokeratin (CK) expression. In this study, HNSCC cell lines were treated with EGF, and CK expression levels were monitored by Western blot analysis. Changes in cellular morphology were documented by fluorescence- and atomic force microscopy. Some of the cell lines demonstrated an EGF-dependent modulation of CK expression levels. Interestingly, regression of some CK subtypes or initial up-regulation followed by downregulation at higher EGF-levels could also be observed in the tested cell lines. Overall, the influence of EGF on CK expression levels appeared variable and cell-type-dependent. Real-time cellular analysis of EGF-treated and -untreated HNSCC cell lines demonstrated a rise over time in cellular impedance. In three of the EGF-treated HNSCC cell lines, this rise was markedly higher than in untreated controls, whereas in one of the cell lines the gain of cellular impedance was paradoxically reduced after EGF treatment, which was found to correlate with changes in cellular morphology rather than with relevant changes in cellular viability or proliferation. After treating HNSCC cells with EGF, CK filaments frequently appeared diffusely distributed throughout the cytoplasm, and in some cases were found in a perinuclear localization, the latter being reminiscent to observations by other groups. In summary, the data points to a possible role of EGFR in modulating HNSCC cell morphology.

Risk of lymph node metastases after en bloc cold steel, en bloc laser-, and piecemeal laser surgical resection of auricular VX2 carcinoma.

There is some controversy in the literature if lymph vessels are enduring sealed during piecemeal CO2 laser surgery of squamous cell carcinomas of the head and neck or a propagation of tumor cells into the lymphatics occurs. The aim of the present study was to analyze the incidence of lymph node and distant metastases after different methods of resection of a VX2 carcinoma in an animal model. A solid auricular VX2 carcinoma was induced in 200 rabbits. Seven days later, an en bloc cold steel (group A), en bloc laser surgical resection with CO2 laser in continuous wave mode with 2 W (group B), or piecemeal laser surgical resection after transection of the tumor with CO2 laser in continuous wave mode with 2 W (group C) or 20 W (group D) was performed. The animals were killed and the incidence of lymph node and distant metastases was compared between the different groups. Of the rabbits, 21.1 % developed lymph node metastases and 10 % pulmonary metastases. The incidence of lymph node metastases was 17.4 % in group A, 20.4 % in group B, 26 % in group C, and 20 % in group D. These differences were not statistically significant. En bloc cold steel, en bloc laser-, or piecemeal laser surgical resections include similar risk of postoperative metastases. Propagation of tumor cells cannot be excluded with certainty by any of these methods.

RNAi-Mediated Knockdown of Cottontail Rabbit Papillomavirus Oncogenes Using Low-Toxicity Lipopolyplexes as a Paradigm to Treat Papillomavirus-Associated Cancers.

The cottontail rabbit papillomavirus (CRPV)-associated VX2 carcinoma of the New Zealand White rabbit serves as a model system for human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCCs). The aim of this study was to evaluate the tumor-inhibiting effect of RNAi-mediated knockdown of the CRPV oncogenes, E6 and E7, using siRNA-loaded lipopolyplexes (LPPs). VX2-carcinoma-derived cells were cultured for up to 150 passages. In addition, CRPV E6 and E7 oncogenes were transiently expressed in COS-7 cells. Efficiency and safety of LPPs were evaluated in both VX2 cells and the COS-7 cell line. Both of these in vitro CRPV systems were validated and characterized by fluorescence microscopy, Western blot, and RT-qPCR. Efficient knockdown of CRPV E6 and E7 was achieved in VX2 cells and COS-7 cells pretransfected with CRPV E6 and E7 expression vectors. Knockdown of CRPV oncogenes in VX2 cells resulted in reduced viability, migration, and proliferation and led to a G0/G1 block in the cell cycle. CRPV E6 and E7 siRNA-loaded LPPs could represent promising therapeutic agents serving as a paradigm for the treatment of papillomavirus-positive cancers and could be of value for the treatment of CRPV-associated diseases in the rabbit such as papillomas and cancers of the skin.

Generation of a Syngeneic Heterozygous ACVRL1(wt/mut) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis.

Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutated. Patients exhibit disturbances in TGF-beta/BMP-dependent angiogenesis and, clinically, often present with severe nosebleeds as well as a reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem cells (iPSCs) and evaluate the effects on TGF-beta- and BMP-related gene expression as well as angiogenesis. The CRISPR/Cas9 knockout of the ACVRL1 gene was carried out in previously characterized wild-type (ACVRL1wt/wt) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1wt/mut) in wild-type (ACVRL1wt/wt) iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the ACVRL1 gene [NG_009549.1:g.13707_13723del; NM_000020.3:c.1137_1153del]. After the generation of embryoid bodies (EBs), endothelial differentiation was induced via adding 4 ng/mL BMP4, 2% B27, and 10 ng/mL VEGF. Endothelial differentiation was monitored via immunocytochemistry. An analysis of 151 TGF-beta/BMP-related genes was performed via RT-qPCR through the use of mRNA derived from single iPS cell cultures as well as endothelial cells derived from EBs after endothelial differentiation. Differential TGF-beta/BMP gene expression was observed between ACVRL1wt/wt and ACVRL1wt/mut iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed ACVRL1 mutant HHT type 2 iPSCs, together with their isogenic wild-type iPSC counterparts, can serve as valuable resources for HHT type 2 in vitro studies.

Intravenous inoculation of a bat-associated rabies virus causes lethal encephalopathy in mice through invasion of the brain via neurosecretory hypothalamic fibers.

The majority of rabies virus (RV) infections are caused by bites or scratches from rabid carnivores or bats. Usually, RV utilizes the retrograde transport within the neuronal network to spread from the infection site to the central nervous system (CNS) where it replicates in neuronal somata and infects other neurons via trans-synaptic spread. We speculate that in addition to the neuronal transport of the virus, hematogenous spread from the site of infection directly to the brain after accidental spill over into the vascular system might represent an alternative way for RV to invade the CNS. So far, it is unknown whether hematogenous spread has any relevance in RV pathogenesis. To determine whether certain RV variants might have the capacity to invade the CNS from the periphery via hematogenous spread, we infected mice either intramuscularly (i.m.) or intravenously (i.v.) with the dog-associated RV DOG4 or the silver-haired bat-associated RV SB. In addition to monitoring the progression of clinical signs of rabies we used immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to follow the spread of the virus from the infection site to the brain. In contrast to i.m. infection where both variants caused a lethal encephalopathy, only i.v. infection with SB resulted in the development of a lethal infection. While qRT-PCR did not reveal major differences in virus loads in spinal cord or brain at different times after i.m. or i.v. infection of SB, immunohistochemical analysis showed that only i.v. administered SB directly infected the forebrain. The earliest affected regions were those hypothalamic nuclei, which are connected by neurosecretory fibers to the circumventricular organs neurohypophysis and median eminence. Our data suggest that hematogenous spread of SB can lead to a fatal encephalopathy through direct retrograde invasion of the CNS at the neurovascular interface of the hypothalamus-hypophysis system. This alternative mode of virus spread has implications for the post exposure prophylaxis of rabies, particularly with silver-haired bat-associated RV.

Sensitivity of Papilloma Virus-Associated Cell Lines to Photodynamic Therapy with Curcumin-Loaded Liposomes.

Photodynamic therapy (PDT) is a minimally invasive therapeutic approach used in the treatment of various medical conditions and cancerous diseases, involving light, a photosensitizing substance, and oxygen. Curcumin, a naturally occurring compound, carries antitumor activities and potentially could be exploited as a photosensitizer in PDT. Only little is known about liposomal-encapsulated curcumin that could help in increasing the efficacy, stability, and bioavailability of this compound. This study investigates the in vitro effects of curcumin-loaded liposomes in combination with PDT. Three papilloma virus-associated cell lines were treated with curcumin-loaded liposomes corresponding to a curcumin concentration of 0-100 µmol/L for 4 h followed by illumination at 457 nm (blue) for 45, 136, and 227 s at a fluence of 220.2 W/m2 (100 mA) corresponding to 1, 3 and 5 J·cm-2. After 24 h, the biological outcome of the treatment was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), SYTO9/PI (propidium iodide), Annexin V-FITC (fluorescein isothiocyanate)/PI, clonogenic survival, and scratch (wound closure) assays. Photoactivation of curcumin-loaded liposomes led to a significant reduction in colony formation and migratory abilities, as well as to an increase in tumor cell death. The results point to the combination of curcumin-loaded liposomes with PDT as a potentially useful tool for the treatment of papillomavirus-associated malignancies.

Aberrant Expression of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in Warthin Tumors.

The Warthin tumor represents the second most frequent benign tumor of the parotid gland and is characterized by the presence of oncocytes rich in structurally and functionally altered mitochondria. Next to its role in metabolism, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also implicated in cellular mitophagy. Immunohistochemistry was carried out on Warthin tumor and normal control (parotid gland with striated ducts) tissues, using anti-GAPDH specific antibodies followed by digital image analysis. Laser capture microdissection was used to isolate the oncocytic tumor cell and normal control striated duct compartments for RNA extraction and qPCR. Warthin tumor oncocytes exhibited a markedly spotted GAPDH staining pattern exhibiting cells with cytoplasmic and nuclear, only nuclear or none GAPDH staining. A significantly lower (p < 0.0001) total GAPDH signal was detected in Warthin tumor oncocytes. Similarly, significantly lower (p < 0.005) GAPDH mRNA levels were seen in oncocytes compared with normal ductal cells. To exclude the possibility of this GAPDH staining pattern being a general feature of oncocytic neoplasms of different organs, we tested a cohort of renal oncocytoma and oncocytic chromophobe carcinoma; none showed this type of staining. The observed progressive GAPDH loss in Warthin tumor oncocytes could be implicated in the pathogenesis of Warthin tumors.

Treatment with ozone/oxygen-pneumoperitoneum results in complete remission of rabbit squamous cell carcinomas.

Head and neck squamous cell carcinomas (HNSCC) represent a group of metastasizing tumors with a high mortality rate in man and animals. Since the biomolecule ozone was found to inhibit growth of various carcinoma cells in vitro we here applied the highly aggressive and lethal VX2 carcinoma HNSCC tumor model of the New Zealand White rabbit to test whether ozone exerts antitumorous effects in vivo. Therapeutic insufflation of medical ozone/oxygen (O(3)/O(2)) gas mixture into the peritoneum (O(3)/O(2)-pneumoperitoneum) at an advanced stage of tumor disease led to a survival rate of 7/14 rabbits. Six of the seven surviving rabbits presented full tumor regression and the absence of local or distant lung metastases. Insufflation of pure oxygen (O(2)) resulted in a survival rate of 3/13 animals accompanied by full tumor remission in 2 of the 3 surviving animals. Of the 14 sham-treated animals only 1 had spontaneous tumor remission and survived. No adverse effects or changes in standard blood parameters were observed after repeated intraperitoneal insufflations of the O(3)/O(2) or O(2) gas. Animals with O(3)/O(2)-induced tumor eradication developed tolerance against reimplantation of the VX2 tumor. This could be reversed by immune suppression with a combination of dexamethasone and cyclosporin A suggesting an antitumorous effect of O(3)/O(2)-mediated activation of the body's own immunosurveillance. Although the exact mechanisms of action are still unclear the present data point to O(3)/O(2)-pneumoperitoneum as a promising new strategy in anticancer therapy.

The Auricular VX2 Carcinoma Is a Suitable Animal Model for Identifying Biomarkers for HNSCC Therapy Response.

BACKGROUND/AIM: The rabbit auricular VX2 carcinoma is an established animal model for human head and neck squamous cell carcinoma (HNSCC). Previously, we observed that intraperitoneal oxidative (O3/O2) stress induced tumor remission. Our aim was to evaluate candidate genes associated with tumor regression. MATERIALS AND METHODS: For identification of tumor remission-related genes, microarray analysis was performed with subsequent validation by polymerase chain reaction (PCR), in situ hybridization, immunohistochemistry and western blot analysis. RESULTS: Microarray analysis indicated a prominent reduction of epidermal growth factor receptor (Egfr, Erbb1) expression levels in regressing tumors. Quantitative PCR confirmed a significant (p<0.005) down-regulation of Erbb1-3 mRNA in regressing VX2 tumors. Histological localization of Erbb1-3 mRNA transcript and protein indicated reduced Erbb gene expression occurring at the level of individual VX2 tumor cells rather than solely being an effect of tumor shrinkage. This study highlights changes in the Erbb gene signature of regressing VX2 carcinomas as a predictor for therapy response. The VX2 carcinoma animal model, therefore, appears suitable for the identification and evaluation of new diagnostic, prognostic and therapeutic biomarkers prior to their application in patients with HNSCC.

Virome Analysis Reveals No Association of Head and Neck Vascular Anomalies with an Active Viral Infection.

BACKGROUND/AIM: Vascular anomalies encompass different vascular malformations [arteriovenous (AVM), lymphatic (LM), venous lymphatic (VLM), venous (VM)] and vascular tumors such as hemangiomas (HA). The pathogenesis of vascular anomalies is still poorly understood. Viral infection was speculated as a possible underlying cause. MATERIALS AND METHODS: A total of 13 human vascular anomalies and three human skin control tissues were used for viral analysis. RNA derived from AVM (n=4) and normal skin control (n=3) tissues was evaluated by RNA sequencing. The Virome Capture Sequencing Platform for Vertebrate Viruses (VirCapSeq-VERT) was deployed on 10 tissues with vascular anomalies (2×AVM, 1×HA, 1×LM, 2×VLM, 4×VM). RESULTS: RNA sequencing did not show any correlation of AVM with viral infection. By deploying VirCapSeq-VERT, no consistent viral association was seen in the tested tissues. CONCLUSION: The analysis does not point to the presence of an active viral infection in vascular anomalies. However, transient earlier viral infections, e.g. during pregnancy, cannot be excluded with this approach.

Evidence for the absence of visceral pain in C57BL6/N mice subjected to therapeutically relevant O3/O2-pneumoperitoneum.

Each different gas that is used to induce a pneumoperitoneum (PP) exhibits individual effects within the peritoneal cavity. This might include adverse effects such as pain and/or inflammatory reactions. The acute effects of ozonized oxygen (O3/O2), a highly oxidative gas mixture, after being insufflated into the peritoneal cavity are analysed in this study. Using the abdominal constriction response ('writhing') assay of chemical nociception in C57BL6/N mice, O3/O2-PP was found not to be associated with visible pain responses and did not alter the c-fos expression in the spinal cord. In addition, mRNA expression levels of the pro-inflammatory cytokines, interleukin (IL)-1ß and IL-6, were found unaltered in the spleen 2 h after insufflation. In conclusion, O3/O2-PP is free of adverse pain and does not trigger inflammatory immune responses.

Treatment of HNSCC cell lines with the EGFR-specific inhibitor cetuximab (Erbitux) results in paradox phosphorylation of tyrosine 1173 in the receptor.

Overexpression of the epidermal growth factor receptor (EGFR, ErbB1, HER1) is frequent in head and neck squamous cell carcinomas (HNSCCs) and correlates with disease progression. Inhibition of EGFR with the kinase inhibitor AG1478 abolished receptor phosphorylation and reduced cell proliferation. However, treatment of HNSCC cells with cetuximab (Erbitux), a monoclonal antibody designed to block the EGFR ligand binding site, led to paradox EGFR activation due to hyperphosphorylation of tyrosine 1173, however, with a concomitant reduction in Erk1/2 phosphorylation levels. No pronounced influence on cell proliferation levels could be observed after treatment with this antibody. Since cetuximab appears able to activate EGFR in HNSCC cell lines, it is necessary to rethink the exact mechanisms by which cetuximab that recently was approved for the treatment of advanced head and neck cancer, inhibits tumor growth.

Efficiency of tazobactam/piperacillin in lethal peritonitis is enhanced after preconditioning of rats with O3/O2-pneumoperitoneum.

Insufflation of ozonized oxygen into the peritoneum (O3/O2-pneumoperitoneum [O3/O2-PP]) of rats reduced the lethality of peritonitis. We evaluated the prophylactic effect of O3/O2-PP combined with tazobactam/piperacillin (TZP) in polymicrobial lethal peritonitis. Wistar rats were conditioned by daily repeated insufflation of ozone for 5 days, and hematologic parameters were determined. Sepsis was induced by i.p. injection of cecal material derived from donor rats. Simultaneously, TZP was applied at a single dosage of 65 mg/kg or at two dosage schedules of 65 mg/kg each at an interval of 1 h. The conditioning effect of O3/O2-PP on the number of blood cells was measured before inoculation of bacteria. The mRNA levels of proinflammatory cytokine IL-lbeta and TNF-alpha were determined at 4 h post infection in spleen and liver by semiquantitative in situ hybridization analysis. Preconditioning of rats by O3/O2-PP enhanced the number of blood leukocytes and granulocytes and increased the survival rate of septic rats up to 33%. The combination of O3/O2-PP and TZP further enhanced the survival rate up to 93%. This effect was accompanied by a reduced amount of IL-1beta and TNF-alpha mRNA in spleen and liver. In contrast, in non-infected animals the combination of O3/O2-PP and TZP enhanced IL-1beta and TNF-alpha mRNA in the spleen and IL-1beta mRNA in liver when compared with TZP- and sham-treated controls. The preconditioning effect of O3/O2-PP seems to support the biological effectiveness of TZP by altering the immune status before and during the onset of sepsis. The combined therapy could be a simple, preoperative intervention for abdominal surgery to reduce postoperative morbidity and mortality.

Constitutive expression of p55TNFR mRNA and mitogen-specific up-regulation of TNF alpha and p75TNFR mRNA in mouse brain.

Serum tumor necrosis factor (TNF) functions as a mediator of the immune-to-brain axis. Numerous TNF receptor-mediated effects on the nervous system are described but the knowledge about the regional and cellular expression of TNF receptor p55TNFR and p75TNFR in vivo is far from being complete. It is unclear whether TNF mediates its neuroimmune effects alone or in combination with other factors, e.g., bacterial mitogens. Here, we investigated the distribution of TNFalpha, p55TNFR, and p75TNFR in normal mouse brain and examined the stimulus-specific effects of lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB) on the expression of the cerebral TNF system. Both mitogens caused enhanced TNFalpha serum levels and induced c-fos mRNA in the paraventricular nucleus but exhibited different effects on the cerebral gene expression of the TNF system. LPS but not SEB rapidly induced TNFalpha mRNA in circumventricular organs (CVOs) followed by spreading of TNFalpha mRNA into brain parenchyma close to the CVOs. The p55TNFR gene was constitutively expressed in many neurons with high levels in brainstem motor nuclei and in neurons of the sensory mesencephalic trigeminal nucleus. Moderate levels of p75TNFR mRNA were seen in single cells scattered throughout the brain in a pattern resembling microglia. Neither LPS nor SEB modulated the p55TNFR gene expression in any region or cell type of the brain, and LPS but not SEB induced p75TNFR mRNA in the CVOs. Thus, enhanced TNF serum levels able to stimulate c-fos mRNA expression in the paraventricular nucleus did not necessarily result in a modulation of the cerebral TNF system.

Induction of epidermal fatty acid binding protein in intravascular monocytes of renal allografts.

During acute rejection of rat renal allografts, numerous activated monocytes accumulate in the vasculature of the graft. These monocytes seem to be involved in allograft destruction. Proinflammatory and effector functions of monocytes and macrophages can be down-regulated by peroxisome proliferators, which are probably transported in the cytoplasm by fatty acid binding proteins (FABPs). We performed renal transplantation in rats in the Dark Agouti-to-Lewis strain combination. Intravascular graft leukocytes were harvested 4 days posttransplantation. Epidermal (E)-FABP mRNA and protein expression were investigated by reverse-transcriptase polymerase chain reaction and immunoblotting, respectively. E-FABP-expressing cells were identified by immunofluorescence. After allogeneic transplantation, intravascular graft leukocytes expressed E-FABP mRNA and protein. In isografts, significantly lower expression levels were observed. E-FABP protein was detected in monocytes expressing ED1 and in alphabeta-T-cell receptor positive T lymphocytes. E-FABP might regulate monocyte activation and may represent a promising target for a therapeutic intervention in allograft rejection.