Hypoestoxide inhibits tumor growth in the mouse CT26 colon tumor model

AIM: To evaluate the effect of the natural diterpenoid, hypoestoxide (HE) on the growth of established colon cancer in mice. METHODS: The CT26.WT mouse colon carcinoma cell line was grown and expanded in vitro. Following the expansion, BALB/c mice were inoculated s.c. with viable tumor cells. After the tumors had established and developed to about 80-90 mm3, the mice were started on chemotherapy by oral administration of HE, 5-fluorouracil (5-FU) or combination. RESULTS: The antiangiogenic HE has previously been shown to inhibit the growth of melanoma in the B16F1 tumor model in C57BL/6 mice. Our results demonstrate that mean volume of tumors in mice treated with oral HE as a single agent or in combination with 5-FU, were significantly smaller (> 60%) than those in vehicle control mice (471.2 mm3 vs 1542.8 mm3, P < 0.01). The significant reductions in tumor burden resulted in pronounced mean survival times (MST) and increased life spans (ILS) in the treated mice. CONCLUSION: These results indicate that HE is an effective chemotherapeutic agent for colorectal cancer in mice and that HE may be used alone or in combination with 5-FU.     

Association of leptin -2548G/A and leptin receptor Q223R polymorphisms with increased risk for oral cancer

Purpose  We investigated the possible association of DNA polymorphisms −2548G/A and Q223R in the leptin (LEP) and leptin receptor (LEPR) genes, respectively, which both affect the amount of circulating cytokine-type hormone leptin, with risk for development of oral cancer. Methods  Polymerase chain reaction-based restriction analysis was performed in DNA samples of 150 patients with oral squamous cell carcinoma (OSCC) and 152 healthy control subjects of equivalent gender, age, and ethnicity (Greeks and Germans). Results  Compared to controls, the homozygous high gene expression genotype A/A of the LEP −2548G/A polymorphism was significantly increased in the subgroups of patients with advanced cancer stages (P = 0.0001; OR 9.0, 95% CI 2.62–30.89), with a positive family history of cancer (P = 0.0346; OR 3.55, 95% CI 1.15–11.01), without tobacco abuse (P = 0.0051; OR 9.69, 95% CI 1.03–91.24), and without alcohol abuse (P = 0.0472; OR 2.16, 95% CI 0.87–5.37). The homozygous low-leptin-binding genotype G/G of the LEPR Q223R polymorphism was strongly associated with an increased risk for OSCC for all patients (P = 0.0028; OR 4.11, 95% CI 1.30–12.97) as well for most of the patient subgroups. Conclusions  The above findings are consistent with the growth-promoting role of leptin in cancer and its induction effect on angiogenesis and metastasis. This is the first study indicating the association of these LEP and LEPR gene polymorphisms with increased risk for OSCC.     

Single horizontal Y-V vermilion plasty including orbicularis oris muscle repair for secondary correction of the whistling defect: A universal technique

Objectives: The present prospective study aimed at objectively evaluating the relevance of a single horizontal Y-V vermilion plasty including orbicularis oris muscle repair for secondary correction of whistling deformities in unilateral as well as bilateral cleft lip cases. Study Design: Ten patients were included in the study (mean age 20.2±6.2 years). The size of the whistling defects was determined on photographs before and 12 months after surgery. Additional surgical procedures like columella lengthening and rhinoplasty were documented. Results: Seven minor and 3 moderate whistling defects were corrected. In 7 patients additional procedures were carried out. The data of the 12 months follow-up showed that the whistling defect was significantly reduced in size (p<0005). In 7 out of 10 patients the result of surgery was rated “good” and in 3 patients “moderate”. Conclusions: The present prospective study is the first one to show on an objective basis that the presented technique allows reducing whistling deformities significantly with good overall results in the majority of the cases. Moreover, the technique can be combined with other corrective procedures like columella lengthening without problems. As a consequence, it is a relevant and universal surgical technique for the correction of whistling defects. Key words:Bilateral cleft lip, unilateral cleft lip, secondary correction, vermillion plasty, whistling defect.     

Computed tomography angiography versus digital subtraction angiography in vascular mapping for planning of microsurgical reconstruction of the mandible

The aim of this work was to compare the potential of computed tomography angiography (CTA) with that of digital subtraction angiography (DSA) in vascular mapping of the external carotid artery (ECA) branches for planning of microvascular reconstructions of the mandible with osteomyocutaneous flaps. In 15 patients CTA and DSA were performed prior to surgery. Selective common carotid angiograms were acquired in two projection for both sides of the neck. Sixteen-slice spiral computed tomography was performed with a dual-phase protocol, using the arterial phase images for 3D CTA reconstruction. Thin-slab maximum intensity projections and volume rendering were employed for postprocessing of CTA data. The detectability of the different ECA branches in CTA and DSA was evaluated by two examiners. No statistically significant differences between CTA and DSA (p=0.097) were found for identifying branches relevant for microsurgery. DSA was superior to CTA if more peripheral ECA branches were included (P=0.030). CTA proved to be a promising alternative to DSA in vascular mapping for planning of microvascular reconstruction of the mandible.     

Implant stability and histomorphometry: a correlation study in human cadavers using stepped cylinder implants

The aim of the present study was to determine the correlation between the primary stability of dental implants placed in edentulous maxillae and mandibles, the bone mineral density and different histomorphometric parameters. After assessing the bone mineral density of the implant sites by computed tomography, 48 stepped cylinder screw implants were installed in four unfixed human maxillae and mandibles of recently deceased people who had bequeathed their bodies to the Anatomic Institute I of the University of Erlangen-Nuremberg for medical-scientific research. Peak insertion torque, Periotest values and resonance frequency analysis were assessed. Subsequently, histologic specimens were prepared, and bone-to-implant contact, the trabecular bone pattern factor (TBPf), the density of trabecular bone (BV/TV) and the height of the cortical passage of the implants were determined. The correlation between the different parameters was calculated statistically. The mean resonance frequency analysis values (maxilla 6130.4+/-363.2 Hz, mandible 6424.5+/-236.2 Hz) did not correlate with the Periotest measurements (maxilla 13.1+/-7.2, mandible -7.9+/-2.1) and peak insertion torque values (maxilla 23.8+/-2.2 N cm, mandible 45.0+/-7.9 N cm) (P=0.280 and 0.193, respectively). Again, no correlations could be found between the resonance frequency analysis, the bone mineral density (maxilla 259.2+/-124.8 mg/cm(3), mandible 349.8+/-113.3 mg/cm3), BV/TV (maxilla 19.7+/-8.8%, mandible 34.3+/-6.0%) and the TBPf (maxilla 2.39+/-1.46 mm-1, mandible -0.84+/-3.27 mm-1) (P=0.140 and 0.602, respectively). However, the resonance frequency analysis values did correlate with bone-to-implant contact of the oral aspect of the specimens (maxilla 12.6+/-6.0%, mandible 35.1+/-5.1%) and with the height of the crestal cortical bone penetrated by the implants in the oral aspect of the implant sites (maxilla 2.1+/-0.7 mm, mandible 5.1+/-3.7 mm) (P=0.024 and 0.002, respectively). The Periotest values showed a correlation with the height of the crestal cortical bone penetrated by the implants in the buccal aspect of the implant sites (maxilla 2.5+/-1.2 mm, mandible 5.4+/-1.2 mm) (P=0.015). The resonance frequency analysis revealed more correlations to the histomorphometric parameters than the Periotest measurements. However, it seems that the noninvasive determination of implant stability has to be improved in order to give a more comprehensive prediction of the bone characteristics of the implant site.     

Contribution of the major secreted yops of Yersinia enterocolitica O:8 to pathogenicity in the mouse infection model

Pathogenic yersiniae (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) harbor a 70-kb virulence plasmid (pYV) that encodes a type III secretion system and a set of at least six effector proteins (YopH, YopO, YopP, YopE, YopM, and YopT) that are injected into the host cell cytoplasm. Yops (Yersinia outer proteins) disturb the dynamics of the cytoskeleton, inhibit phagocytosis by macrophages, and downregulate the production of proinflammatory cytokines, which makes it possible for yersiniae to multiply extracellularly in lymphoid tissue. Y. enterocolitica serotype O:8 belongs to the highly mouse-pathogenic group of yersiniae in contrast to Y. enterocolitica serotype O:9. However, there has been no systematic study of the contribution of Yops to the pathogenicity of Y. enterocolitica O:8 in mice. We generated a set of yop gene deletion mutants of Y. enterocolitica O:8 by using the novel Red cloning procedure. We subsequently analyzed the contribution of yopH, -O, -P, -E, -M, -T, and -Q deletions to pathogenicity after oral and intravenous infection of mice. Here we showed for the first time that a DeltayopT deletion mutant colonizes mouse tissues to a greater extent than the parental strain. The DeltayopO, DeltayopP, and DeltayopE mutants were only slightly attenuated after oral infection since they were still able to colonize the spleen and liver and cause systemic infection. The DeltayopO mutant was lethal for mice, whereas DeltayopP and DeltayopE mutants were successfully eliminated from the spleen and liver 2 weeks after infection. In contrast the DeltayopH, DeltayopM, and DeltayopQ mutants were highly attenuated and not able to colonize the spleen and liver on any of the days tested. The DeltayopH, DeltayopO, DeltayopP, DeltayopE, DeltayopM, and DeltayopQ mutants had only modest defects in the colonization of the small intestine and Peyer's patches. The DeltayopE mutant was eliminated from the small intestine 3 weeks after infection, whereas the DeltayopH, DeltayopP, DeltayopM, and DeltayopQ mutants continued to colonize the small intestine at this time.     

Concomitant cytosolic delivery of two immunodominant listerial antigens by Salmonella enterica serovar typhimurium confers superior protection against murine listeriosis

During its interaction with host cells, Salmonella enterica serovar Typhimurium employs a type III secretion system for cytosolic targeting of virulence factors. This protein translocation mechanism is a useful tool for heterologous antigen delivery by attenuated Salmonella vaccine carrier strains. In the present study, we used the Yersinia outer protein E (YopE) as a carrier molecule for Salmonella type III-dependent cytosolic delivery of the immunodominant CD8 T-cell antigens listeriolysin O (LLO) and p60 of Listeria monocytogenes. It is shown that concomitant translocation of hybrid YopE/LLO and YopE/p60 proteins by Salmonella led to antigen presentation and CD8 T-cell priming efficacies comparable to those of translocation of single listerial antigens. However, simultaneous translocation of LLO and p60 significantly surpassed single cytosolic antigen delivery in the ability to protect against LISTERIA: For the first time, this study demonstrates that concomitant expression of two independent antigens via the same recombinant plasmid leads to superior protection against a challenge with an intracellular bacterial pathogen. In conclusion, these findings emphasize the versatility of Salmonella type III-mediated heterologous antigen delivery for the induction of cytotoxic T-lymphocyte-mediated immunity.     

Salmonella pathogenicity island 2-mediated overexpression of chimeric SspH2 proteins for simultaneous induction of antigen-specific CD4 and CD8 T cells

Salmonella enterica serovar Typhimurium employs two different type III secretion systems (TTSS) encoded within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) for targeting of effector proteins into the cytosol of eukaryotic cells during different stages of the infection cycle. The SPI1 TTSS translocates virulence factors across the plasma membrane when the bacterium initially contacts the host cell. In contrast, the SPI2 TTSS functions to translocate proteins across the membrane of the Salmonella-containing vacuole and promotes intracellular survival and replication. The aim of the present study was to directly compare the potentials of SPI1 and SPI2 type III effector proteins to act as carrier molecules for a heterologous antigen. The p60 protein of Listeria monocytogenes was used as a model antigen to construct chimeric SopE2 (SPI1), SifA (SPI2), and SspH2 (SPI2) proteins. SPI1- and SPI2-dependent up- and down-regulation of hybrid gene expression led to sequential translocation of p60 fusion proteins into the cytosol of Salmonella-infected macrophages. Mice orally immunized with recombinant Salmonella strains expressing these hybrid proteins revealed comparable numbers of p60-specific CD8 T cells. However, only overexpression of translocated SspH2/p60 from a medium-copy-number vector induced simultaneous antigen-specific CD4 and CD8 T-cell responses, suggesting that SspH2 is an attractive carrier molecule for foreign-protein delivery.