Nikolaus Freudenberg

Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity

Allergic contact hypersensitivity (CHS) is a T cell–mediated inflammatory skin disease. Interleukin (IL)-12 is considered to be important in the generation of the allergen-specific T cell response. Loss of IL-12 function in IL-12Rβ2–deficient mice, however, did not ameliorate the allergic immune response, suggesting alternate IL-12–independent pathways in the induction of CHS. Because exposure to contact allergens always takes place in the presence of microbial skin flora, we investigated the potential role of Toll-like receptors (TLRs) in the induction of CHS. Using mice deficient in TLR4, the receptor for bacterial lipopolysaccharide (LPS), IL-12 receptor (R) β2, or both, we show that the concomitant absence of TLR4 and IL-12Rβ2, but not the absence of TLR4 or IL-12Rβ2 alone, prevented DC-mediated sensitization, generation of effector T cells, and the subsequent CHS response to 2,4,6-trinitro-1-chlorobenzene (TNCB), oxazolone, and fluorescein isothiocyanate. Introduction of the TLR4 transgene into the TLR4/IL-12Rβ2 mutant restored the CHS inducibility, showing a requirement for TLR4 in IL-12–independent CHS induction. Furthermore, the concomitant absence of TLR2 and TLR4 prevented the induction of CHS to TNCB in IL-12–competent mice. Finally, CHS was inducible in germ-free wild-type and IL-12Rβ2–deficient mice, but not in germ-free TLR4/IL-12Rβ2 double deficient mice, suggesting that the necessary TLR activation may proceed via endogenous ligands.

Requirement for TLR9 in the immunomodulatory activity of Propionibacterium acnes.

Propionibacterium acnes (formerly Corynebacterium parvum) is part of the human flora and, as such, is associated with several human pathologies. It possesses strong immunomodulatory activities, which makes this bacterium interesting for prophylactic and therapeutic vaccination. The bacterial component(s) and the host receptor(s) involved in the induction of these activities are poorly understood. We show in this study that TLR9 is crucial in generating the characteristic effects of killed P. acnes priming in the spleen, such as extramedullary hemopoiesis and organ enlargement, and granuloma formation in the liver. Furthermore, the ability to overproduce TNF-α and IFN-γ in response to LPS, lipid A, synthetic lipopeptide Pam3CysK4, or whole killed bacteria was present in P. acnes-primed wild-type, but not TLR9−/−, mice. Finally, P. acnes priming failed to induce enhanced resistance to murine typhoid fever in TLR9−/− mice. Thus, TLR9 plays an essential role in the induction of immunomodulatory effects by P. acnes. Because IFN-γ is a key mediator of these effects, and enhanced IFN-γ mRNA expression was absent in spleen and liver of P. acnes-primed TLR9−/− mice, we conclude that TLR9 is required for the induction of IFN-γ by P. acnes.

Performance of the Aptima High-Risk Human Papillomavirus mRNA Assay in a Referral Population in Comparison with Hybrid Capture 2 and Cytology

ABSTRACT This study compared the Aptima human papillomavirus (HPV) (AHPV; Gen-Probe Incorporated) assay, which detects E6/E7 mRNA from 14 high-risk types, the Hybrid Capture 2 HPV DNA (HC2; Qiagen Incorporated) test, and repeat cytology for their ability to detect high-grade cervical lesions (cervical intraepithelial neoplasia grade 2+ [CIN2+]) in women referred to colposcopy due to an abnormal Papanicolaou (Pap) smear. A total of 424 clinical specimens, stored in liquid-based cytology (LBC) vials at room temperature for up to 3 years, were tested by repeat cytology, the AHPV assay, and the HC2 test. Assay results were compared to each other and to histology results. The overall agreement between the AHPV assay and the HC2 test was 88.4%. The sensitivity (specificity) of cytology, the HC2 test, and the AHPV assay for the detection of CIN2+ was 84.9% (66.3%), 91.3% (61.0%), and 91.7% (75.0%) and for the detection of CIN3+ was 93.9% (54.4%), 95.7% (46.0%), and 98.2% (56.3%), respectively. Of the disease-positive specimens containing high-risk HPV (HR HPV) DNA as determined by Linear Array (Roche Diagnostics), the AHPV assay missed 3 CIN2 and 1 microfocal CIN3 specimen, while the HC2 test missed 6 CIN2, 4 CIN3, and 1 cervical carcinoma specimen. The AHPV assay had a sensitivity similar to but a specificity significantly higher (P < 0.0001) than the HC2 test for the detection of CIN2+. The AHPV assay was significantly more sensitive (P = 0.0041) and significantly more specific (P = 0.0163) than cytology for the detection of disease (CIN2+).

Performance of p16INK4a-cytology, HPV mRNA, and HPV DNA testing to identify high grade cervical dysplasia in women with abnormal screening results

OBJECTIVE The prognostic value of dysplastic lesions of the uterine cervix cannot be adequately determined by Pap cytology alone. Detection of HPV DNA increases the diagnostic sensitivity. However, due to the very high prevalence of transient HPV infections, HPV DNA testing suffers from poor diagnostic specificity. Biomarkers that highlight the shift from self limited transient to potentially dangerous transforming HPV infections may improve the accuracy of cervical cancer screening. We evaluated HPV E6/E7 mRNA detection (APTIMA), p16(INK4a)-immunocytology (CINtec), and HPV DNA testing (HC2) to identify women with high grade cervical neoplasia in a disease-enriched cross-sectional cohort. METHODS Liquid based cytology specimens were collected from 275 patients. All assays were performed from these vials. Detection rates of each test were evaluated against conventional H&E based histopathology alone and stratified by p16(INK4a)-immunohistochemistry (IHC). RESULTS All assays yielded a high sensitivity for the detection of CIN3+ (96.4% (95% CI, 90.4-98.8) for HC2, 95.5% (89.2-98.3) for APTIMA and CINtec) and CIN2+ (91.5% (85.8-95.1) for HC2, 88.4% (82.3-92.7) for APTIMA, 86.6% (80.2-91.2) for CINtec). The specificity to detect high grade dysplasia was highest for CINtec p16(INK4a)-cytology (60.6% (52.7-68.0) in CIN3+ and 74.8% (65.5-82.3) in CIN2+), followed by APTIMA (56.4% (48.4-64.0) in CIN3+ and 71.2% (61.7-79.2) in CIN2+) and HC2 (49.1% (41.3-56.9) in CIN3+ and 63.4% (53.7-72.1) in CIN2+). All tests had higher sensitivity using p16(INK4a)-IHC-positive CIN2+ lesions as endpoint. CONCLUSIONS Biomarkers that detect HPV induced dysplastic changes in the transforming stage are promising tools to overcome the current limitations of cervical cancer screening.

Comparison of the Sensitivity of Sputum and Brush Cytology in the Diagnosis of Lung Carcinomas

OBJECTIVE To describe the role of sputum and brush cytology in the diagnosis of lung carcinoma and to elucidate the influence of tumor location, histologic tumor type and stage on the sensitivity of both methods. STUDY DESIGN Retrospective and performed on 415 lung cancer patients. Two hundred of them were investigated only by sputum collection, 119 only by brushing and 96 by both methods. RESULTS The overall sensitivity of the sputum technique was 0.403 and that of the brush method 0.500, while a combination of both showed a sensitivity of 0.640. The diagnostic yield depended on tumor location, histologic tumor type and stage. Sputum specimens were most valuable in the detection of early and peripheral carcinomas, whereas brushing was superior in finding more advanced and centrally located malignancies. Regarding tumor type, squamous cell carcinomas were diagnosed to the greatest extent by both methods. CONCLUSION A complementary role of both cytologic techniques can be postulated by our data as well as by a literature review.

Infiltration of lung carcinomas with macrophages of the 27E10-positive phenotype

In order to gain insight into the role of macrophages in human lung carcinomas, we investigated material from 35 lung carcinomas and 5 healthy lungs with 4 different antibodies (CD68, MRP8, MRP14, 27E10) recognizing different macrophage subtypes. Infiltration with CD68-positive macrophages was highest and comparable in healthy lungs and lung carcinomas. Compared to healthy lungs, the infiltration of MRP8- and MRP14-positive macrophages was reduced in lung carcinomas while the number of 27E10-positive cells was enhanced. No difference in the infiltration of macrophages was observed between the different histological subtypes of carcinomas such as squamous carcinoma, small lung carcinoma, adenocarcinoma and bronchio-alveolar carcinoma. Furthermore, we present a highly suitable technique for the isolation and enrichment of macrophages from human lung carcinomas resulting in a 5-10 fold enrichment and a yield of e.g. 2-3 x 10(6) 27E10-positive macrophages/g tumor biopsy. Together with the recent findings that 27E10-positive macrophages are prevalent in early acute inflammation and release cytotoxic mediators and to inhibit tumor cell proliferation our findings suggest that 27E10-positive macrophages may play a role in antitumor cytotoxicity in human lung carcinomas.

Investigations into the origin of mouse liver sinusoidal cells

The possibility that liver sinusoidal cells are derived from the bone-marrow was investigated in chimeric mice. H2k-positive bone-marrow cells from F1 (B10.BR × B10.D2) hybrid mice were transplanted into irradiated H2k-negative parental mice (B10.D2), and the liver examined immunohistochemically for the presence of H2k-positive cells, with the help of an anti-H2k monoclonal antibody. With the passage of time (from the fifth week onwards), increasing numbers of transplanted bone-marrow cells enter the liver sinusoids, undergo alteration in their shape, and remain there, probably replacing sinusoidal lining cells. DNA-synthesising cells in the sinusoids were observed, suggesting, in addition, local cell proliferation. The replacement of sinusoidal cells from bone-marrow was greatly accelerated after liver damage had been induced by sublethal doses of endotoxin (LPS), and proliferation was also enhanced after treatment with LPS. These results strongly suggest that the bone-marrow participates in the replacement of liver sinusoidal cells.

Surfactant Protein A Detection in Primary Pulmonary Adenocarcinoma without Bronchioloalveolar Pattern

Background: Immunohistochemical studies in human lung carcinoma reported positive staining of tumor cells for surfactant protein A (SP-A), especially in peripheral airway cell carcinoma, which include bronchioloalveolar carcinoma and in some reports also papillary subtypes. Objective: The purpose of this study was to determine the SP-A expression in tumor cells of lung adenocarcinoma without a bronchioloalveolar pattern, classified according to the WHO. Methods: In total, 169 primary adenocarcinomas of the lung (109 acinar, 32 solid with mucin, 24 papillary and 4 mucinous) were examined by immunohistochemistry for SP-A expression. Results: Twenty-five percent of acinar, 38% of papillary and 3% of solid adenocarcinoma with mucin showed a positive intracytoplasmic SP-A reaction of the tumor cells. None of the mucinous adenocarcinomas stained for SP-A. This study included the largest number of acinar adenocarcinomas and solid adenocarcinomas with mucin studied for SP-A. We clearly demonstrated that also primary lung adenocarcinoma without a bronchioloalveolar pattern can express SP-A. A positive staining of hyperplastic type II cells surrounding the tumors or entrapped in the tumor could clearly be differentiated from the SP-A-positive stain of tumor cells. Conclusion: These results support the theory that SP-A-producing cells may generate not only bronchioloalveolar and papillary carcinoma, but also other subtypes of lung adenocarcinoma.

Experimental gluing of the bronchial stump after pneumonectomy in rats.

OBJECTIVE The objective of this experimental study was to evaluate the usefulness of gelatin-resorcinol-dialdehyde adhesive in sutureless closure of bronchial stumps. METHODS In 40 male Wistar rats bronchial stumps after left-sided pneumonectomy were closed by gluing with gelatin-resorcinol-dialdehyde adhesive. For macroscopic and microscopic examination four animals were sacrificed on postoperative days 2, 7 and 14 each, 14 animals on postoperative days 28 and 120 each. RESULTS On macroscopic examination the gelatin-resorcinol-dialdehyde adhesive proved in all cases effective in tight bronchial stump closure. The adhesive did not cause local infection or necrosis of the bronchial stump nor other intrathoracic inflammatory complications. All animals survived and made an uncomplicated postoperative recovery. Microscopic examination revealed that the gelatin-resorcinol-dialdehyde adhesive initially evoked an acute inflammatory response with polymorphonuclear neutrophils predominating. After an intermediate stage characterized by a granulomatous reaction and resorption of the adhesive by multinucleated giant cells, 120 days postoperatively the bronchial stumps at the sites of previous gluing showed a regular fibrous scar tissue without inflammatory cells. CONCLUSION The gelatin-resorcinol-dialdehyde adhesive showed effective in closing bronchial stumps after pneumonectomy in rats. The clinical extrapolation of this effect to thoracic surgical patients is uncertain at this time.

Reactive alveolar epithelium in chondroid hamartoma of the lung.

OBJECTIVE To determine the morphologic characteristics of the nonciliated epithelium found in chondroid hamartoma of the lung. STUDY DESIGN The morphologic characteristics and immunohistochemical reaction for surfactant protein A of the nonciliated epithelium in chondroid hamartoma of the lung was studied by immunohistochemistry. Alveolar epithelium in normal lung tissue and lung tissue surrounding primary lung cancer or metastatic lung lesions was used as a control. RESULTS In all cases, the nonciliated epithelium in chondroid hamartoma showed the morphologic criteria of hyperplastic alveolar type II cells and a very strong positive surfactant protein A reaction in the cytoplasm when compared with alveolar epithelium of the normal lung. Similar hyperplastic type II cells were also found in the alveolar lung around metastatic or primary lung tumors. CONCLUSION These findings may indicate that the nonciliated cells found in chondroid hamartoma of the lung are hyperplastic type II cells. This suggests that the alveolar epithelium found in chondroid hamartoma of the lung is a secondary reaction around the hamartoma and not a primary component of the lesion.