Malignant and non-malignant lung tissue areas are differentially populated by natural killer cells and regulatory T cells in non-small cell lung cancer.

Even though the lung represents a special immune compartment with the capacity of a high inflammatory response, ineffective anti-tumour immunity is common in lung-associated malignancies. We asked whether a differential composition of the immune cell infiltrate in malignant (MLTAs) and non-malignant lung tissue areas (N-MLTAs) exists and might potentially contribute to this effect. We performed a comparative analysis of immune cells residing in MLTAs and N-MLTAs of non-small cell lung cancer (NSCLC) patients. To this end, we used immunophenotyping and functional analyses on directly isolated immune cells and tissue arrays on archived paraffin-embedded specimens. A strong T cell infiltration was prominent in both tissue compartments whereas CD4(+)CD25(+)CD127(-) T regulatory cells were present in MLTAs only. Nonetheless, concurrent functional ex vivo T cell analyses revealed no significant difference between T cells of MLTA and N-MLTA, suggesting that tumour-infiltrating T cells were not functionally impaired. Interestingly, T cell infiltration was less pronounced in specimens with a high neutrophilic infiltrate. NK cell infiltration was strikingly heterogenous between MLTA and N-MLTA. While NK cells were almost absent in the malignant tissue regions, non-malignant counterparts were selectively populated by NK cells and those NK cells showed strong cytotoxic activity ex vivo. We report that malignant and non-malignant tissue areas in NSCLC are selectively infiltrated by certain immune cell types with NK cells being displaced from the tumour tissue. These phenomena have important implications for tumour immunology of NSCLC and should be considered for the development of future immunologic intervention therapies.

[General surgery under discussion. From the viewpoint of thoracic surgery]. / Allgemeinchirurgie in der Diskussion. Aus Sicht der Thoraxchirurgie.

The development of surgery in Germany was correct and to the point. General surgery was the field from which all specialization grew. Surgical fields today must comply with scientific demands and medical progress. They can remain successful only if the various branches respond to developments in respect to minimum capacities, procedural and structural conditions, and specialized education and training. "General surgery" as a basic foundation provides an important cross-section of the eight-pronged model of the various specializations. As such it is able to deliver effectively a broad spectrum of good patient care. The network of thoracal surgical centers guarantees sufficient and efficient emergent care, because the more beams, the stronger is the roof. At the same time we must remain responsive to developments in the European Union as a whole. Determination and a progressive view by all surgical societies allow the kind of structuring that will guarantee the future of general surgery. We must inspire our new trainees!

Lung fibroblasts from patients with emphysema show markers of senescence in vitro.

BACKGROUND: The loss of alveolar walls is a hallmark of emphysema. As fibroblasts play an important role in the maintenance of alveolar structure, a change in fibroblast phenotype could be involved in the pathogenesis of this disease. In a previous study we found a reduced in vitro proliferation rate and number of population doublings of parenchymal lung fibroblasts from patients with emphysema and we hypothesized that these findings could be related to a premature cellular aging of these cells. In this study, we therefore compared cellular senescence markers and expression of respective genes between lung fibroblasts from patients with emphysema and control patients without COPD. METHODS: Primary lung fibroblasts were obtained from 13 patients with moderate to severe lung emphysema (E) and 15 controls (C) undergoing surgery for lung tumor resection or volume reduction (n = 2). Fibroblasts (8E/9C) were stained for senescence-associated beta-galactosidase (SA-beta-Gal). In independent cultures, DNA from lung fibroblasts (7E/8C) was assessed for mean telomere length. Two exploratory 12 k cDNA microarrays were used to assess gene expression in pooled fibroblasts (3E/3C). Subsequently, expression of selected genes was evaluated by quantitative PCR (qPCR) in fibroblasts of individual patients (10E/9C) and protein concentration was analyzed in the cell culture supernatant. RESULTS: The median (quartiles) percentage of fibroblasts positive for SA-beta-Gal was 4.4 (3.2;4.7) % in controls and 16.0 (10.0;24.8) % in emphysema (p = 0.001), while telomere length was not different. Among the candidates for differentially expressed genes in the array (factor > or = 3), 15 were upregulated and 121 downregulated in emphysema. qPCR confirmed the upregulation of insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-rP1 (p = 0.029, p = 0.0002), while expression of IGFBP-5, -rP2 (CTGF), -rP4 (Cyr61), FOSL1, LOXL2, OAZ1 and CDK4 was not different between groups. In line with the gene expression we found increased cell culture supernatant concentrations of IGFBP-3 (p = 0.006) in emphysema. CONCLUSION: These data support the hypothesis that premature aging of lung fibroblasts occurs in emphysema, via a telomere-independent mechanism. The upregulation of the senescence-associated IGFBP-3 and -rP1 in emphysema suggests that inhibition of the action of insulin and insulin-like growth factors could be involved in the reduced in vitro-proliferation rate.

[Intensive care treatment following thoracic surgery]. / Intensivmedizin aus Sicht des Thoraxchirurgen.

Intensive care in thoracic surgery requires highly specialised abilities and knowledge in addition to usual intensive care standards. The operative chest intensivist must be experienced in special surgical methods, interventional bronchology, and tube management. Cooperating with chest physicians and anaesthesiologists, he takes the central position in intensive therapy. This is described in detail and explained by examples. Furthermore, aspects of education in this specialty will be emphasised.

The effect of selective and non-selective phosphodiesterase inhibitors on allergen- and leukotriene C(4)-induced contractions in passively sensitized human airways.

Non-selective inhibitors of cyclic nucleotide phosphodiesterase (PDE) block allergen-induced contraction of passively sensitized human airways in vitro by a dual mechanism involving a direct relaxant effect on smooth muscle and inhibition of histamine and cysteinyl leukotriene (LT) release from airways. We investigated the effects of non-selective PDE inhibitors and selective inhibitors of PDE3 and PDE4 in order to determine the involvement of PDE isoenzymes in the suppression of allergic bronchoconstriction. Macroscopically normal airways from 76 patients were sensitized with IgE-rich sera (>250 u ml(-1)) containing specific antibodies against allergen (Dermatophagoides farinae). Contractile responses of bronchial rings were assessed using standard organ bath techniques. Passive sensitization caused increased contractile responses to allergen, histamine and LTC(4). Non-selective PDE inhibitors (theophylline, 3-isobutyl-1-methylxanthine [IBMX]), a PDE3-selective inhibitor (motapizone), PDE4-selective inhibitors (RP73401, rolipram, AWD 12-281) and a mixed PDE3/4 inhibitor (zardaverine) all significantly relaxed inherent bronchial tone at resting tension and to a similar degree. Theophylline, IBMX, zardaverine and the combination of motapizone and RP73401 inhibited the contractile responses to allergen and LTC(4). Pre-treatment with motapizone, RP73401, rolipram or the methylxanthine adenosine receptor antagonist, 8-phenyltheophylline, did not significantly decrease responses to either allergen or LTC(4). We conclude that combined inhibition of PDE3 and PDE4, but not selective inhibition of either isoenzyme or antagonism of adenosine receptors, is effective in suppressing allergen-induced contractions of passively sensitized human airways. The relationship between allergen- and LTC(4)-induced responses suggests that PDE inhibitors with PDE3 and PDE4 selectivity are likely to act in part through inhibition of mediator release and not simply through direct relaxant actions on airway smooth muscle.

Preferential histiotypic expression of CD44-isoforms in human lung cancer.

The CD44 transmembrane glycoprotein is expressed in most adult tissues and in the majority of neoplasias. Due to alternative splicing, this cell adhesion molecule exists in multiple isoforms some of which have been associated with specific types of tumours as well as with increased tumour metastasis. In this study, we have looked at the level and type of CD44 expression in lung cancer which represents a histologically heterogenous form of cancer composed of small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), the latter subgroup comprising adenocarcinoma (ADC), bronchio-alveolar carcinoma (BAC), large cell carcinoma (LCC), and squamous cell carcinoma (SCC). We analysed 20 lung cancer cell lines and 64 primary tumours by RT-PCR and immunohistochemical detection of the CD44 standard and variant protein isoforms. Our results suggest that (i) CD44 is expressed in all histologically distinct subsets of lung cancer with a tendency SCC > BAC > ADC > LCC > SCLC, (ii) expression of the CD44 isoforms v5, v7, v8, and, most notably that of CD44 exon v6, strongly correlates with tumours of squamous cell and bronchio-alveolar carcinoma origin, tumours which commonly exhibit a comparatively low metastasizing potential, and (iii) the expression of CD44 isoforms is independent from the tumour size and lymph node status at surgery, the proliferative status of the tumour cell population (Ki67 antigen expression) and the histopathological grading (G1 to G3). Only non-differentiated tumours (G4), which were restricted to SCLC and LCC samples revealed markedly reduced CD44 standard and isoform antigen. In conclusion, our data point to a clear histiotype-related pattern of CD44 variant expression preferentially that of CD44v6 in SCC and BAC.

Absence of high-affinity binding sites for beta-adrenergic blockers and lack of adenyl cyclase stimulation to beta-adrenergic stimulators in most normal and adenomatous human thyroid tissues.

To determine whether the beta-blocking drug propranolol had any physiologic effect on normal (n = 14) and adenomatous (n = 15) human thyroid tissues, experiments were performed to study the binding of the beta-blockers 125I-iodocyanopindolol (125I-ICYP) and 125I-iodohydroxybenzylpindolol (125I-IHYP) and the stimulation of adenyl cyclase (AC) by isoproterenol. 125I-ICYP and 125I-IHYP failed to show high-affinity binding in 27 of 29 specimens, whereas two (one normal and one adenomatous) thyroid tissues demonstrated high-affinity binding (Kd 5.5 +/- 1 X 10(-9) M) for 125I-ICYP. Thyroid-stimulating hormone (0.3 IU/ml), guanosine triphosphate (10(-4) M), and Gpp (NH)p(10(-4) M) stimulated AC in all thyroid tissues, although in two tissues (normal) Gpp (NH)p failed to cause a significant increase. Isoproterenol (10(-4) M), in contrast, had no effect on basal AC activity or on guanosine triphosphate, and Gpp (NH) p stimulated AC activity in 26 of the 29 thyroid tissues. In one of the two tissues that increased AC in response to isoproterenol, the beta-blocking drugs propranolol hydrochloride, bunitrolol hydrochloride, and tolilprolol hydrochloride decreased AC stimulation to isoproterenol at concentrations of 10(-6) M (p less than 0.05). Higher concentrations of propranolol (10(-4) - 10(-2) M) decreased AC stimulation to thyroid-stimulating hormone (p less than 0.01), not only in this responsive tissue but also in tissues that failed to demonstrate high-affinity binding for 125I-ICYP and AC stimulation to isoproterenol (p less than 0.01). Thus most normal and adenomatous human thyroid tissues lack beta-receptors and a functioning beta-receptor AC system. High concentrations of propranolol in vitro decreased AC response by thyroid-stimulating hormone, but this is probably a nonreceptor-mediated effect.

Nerve growth factor (NGF) sensitizes human medullary thyroid carcinoma (hMTC) cells for cytostatic therapy in vitro.

Medullary thyroid carcinoma (hMTC) cells were established from nine patients with MTC disease to initiate a new approach of adjuvant medical therapy in these patients. We measured calcitonin (CT) secretion, DNA synthesis, and cell proliferation in vitro and their response to various substances. Nerve growth factor (NGF) (0.01 to 10 micrograms/ml), glucagon (0.01 to 100 micrograms/ml), and isoproterenol (4 to 500 micrograms/ml) stimulated CT secretion and DNA synthesis in hMTC cells. Other substances, calcium (1.0 to 15 mmol), pentagastrin (1.0 to 50 mumol), dibutyryl-cyclic-adenosine-monophosphate (1.0 to 100 mumol), and phorbol ester TPA (1.0 to 100 nmol), stimulated CT secretion but not DNA synthesis. In addition, NGF enhanced cell proliferation of hMTC cells 2- to 3- fold and caused an increased sensitivity of these cells for chemotherapy in vitro. Thus 0.5 microgram/ml doxorubicin (half-maximal effective dose) induced a cell death rate of up to 32.8%, which was enhanced by preincubation with NGF to 68.1% (1.0 microgram/ml, NGF) and to 100% (10.0 micrograms/ml, NGF), respectively. Pulsative stimulation of APUD cell carcinomas with NGF may therefore improve the response rate of these tumors to chemotherapy, which would be of significant clinical importance for patients with residual postoperative MTC tissue.

The effect of thyrotropin and cAMP on DNA synthesis and cell growth of human thyrocytes in monolayer culture.

Monolayer cultures of human thyrocytes from normal tissue (n = 10), and adenomas (n = 7), differentiated (n = 4), poorly differentiated (n = 2), and undifferentiated (n = 3) thyroid cancers were established to assess the significance of thyrotropin (TSH) and cAMP (adenosine 3',5'-cyclic monophosphate) on cell growth and DNA (deoxyribonucleic acid) synthesis. Cell growth of thyrocytes from normal and adenomatous tissues increased more rapidly (p less than 0.01) after TSH (0.1 IU/ml) was added but was unaffected by cAMP (10(-4) mol/L). In these cells, TSH also enhanced DNA synthesis twofold to twelvefold (p less than 0.01). The adenylate cyclase (AC) inhibitor, 2',3' dideoxyadenosine (ddA), increased DNA synthesis 1.3 to 6 times at a concentration of 2 X 10(-4) mol, whereas the membrane/passable cAMP analogue, dibutyryl-cAMP, and the AC stimulator, forskolin, failed to show any effect on DNA synthesis up to a concentration of 10(-5) mol/L (p less than NS). When administered simultaneously, TSH (1/2 maximum) and ddA (20 mumol) had no cumulative effect on DNA synthesis (p = NS). TSH stimulation in cancerous thyroid tissue (n = 11) demonstrated a lack of TSH response in seven of 11 monolayer cultures with no apparent correlation to cancer differentiation, patient age, or sex. Thus TSH was demonstrated to stimulate DNA synthesis and cell growth of human thyrocytes in monolayer cultures independent of the AC system. However, the TSH effect on cell growth and DNA synthesis was unpredictable in thyrocytes from cancerous tissues.

The effect of the vasoactive intestinal polypeptide agonist Ro 25-1553 on induced tone in isolated human airways and pulmonary artery.

Ro 25-1553 is a metabolically stable analogue of endogenous vasoactive intestinal polypeptide (VIP). This compound is a potent bronchodilator in vitro as well as in vivo. Moreover, Ro 25-1553 has been shown to be highly selective of the VPAC2 receptor. We assessed the effect of Ro 25-1553 on isolated human bronchi and pulmonary arteries in vitro. Macroscopically normal human airways and pulmonary arteries were obtained from patients undergoing surgery for lung cancer. The relaxing capability of Ro 25-1553 on bronchial and pulmonary artery tone was measured using standard techniques. Bronchial rings were pre-contracted with 0.1 mM histamine, and tone in pulmonary artery rings was induced with 10 microM PGF2alpha. Increasing concentrations of Ro 25-1553 within a range of 1 pM to 10 microM were added and isometric tension changes were recorded. Ro 25-1553 caused a concentration-dependent relaxation of airway and pulmonary artery preparations, with an EC50 of approximately 10 nM and a maximal relaxation of 70%-75% of the induced tone. The presence of VPAC2 receptors in the two tissues, though low in density, was confirmed by in situ hybridization, immunocytochemistry and ligand binding. These findings indicate that the VIP analogue Ro 25-1553 may be useful in the treatment of asthma and/or chronic obstructive pulmonary diseases.

The effect of the enantiomers of formoterol on inherent and induced tone in guinea-pig trachea and human bronchus.

The long-acting beta2-adrenoceptor agonist formoterol is, like all other members of this class of drugs, used as a racemate in the clinic. While the effects of the individual enantiomers have been studied on airway smooth muscle from guinea pig, comparable data on human bronchial smooth muscle are scanty or absent. Therefore, we compared the effects of the enantiomers of formoterol on inherent and induced tone in isolated human bronchi with that on guinea-pig trachea in vitro. The human bronchi either were studied under resting tension conditions or were precontracted with 10 microM carbachol or 0.1 mM histamine. The guinea-pig trachea was precontracted with 0.01, 0.1 or 1 microM carbachol. The racemate and (R,R)-formoterol caused a concentration-dependent relaxation of all preparations with an EC50 of about 1 nM. In the guinea-pig trachea, the concentration-effect curve for formoterol was moved to the right in response to an increased concentration of carbachol. In both human bronchus and guinea-pig trachea, (S,S)-formoterol was more than 1,000 times less potent than (R,R)-formoterol. Thus the relaxing effect of formoterol in human airways as well as in guinea-pig trachea was shown to lie with the (R,R)-enantiomer. Notably, (S,S)-formoterol did not exert any contractile effects within the tested concentration range in either airway preparation. Therefore, we conclude that with regard to relaxant effects the pure (R,R)-enantiomer of formoterol does not offer a benefit over the racemate.

Diagnostic and therapeutic strategy in malignant pleural mesothelioma.

We retrospectively analysed 301 patients with diffuse malignant pleural mesothelioma (235 male, 66 female; median age 59 years). Prognosis depended significantly on patient age, evidence of pain, loss of weight, tumour cell type, stage, local and distant metastasis, involvement of peritoneum and surgical treatment. The overall median survival rate was 238 days, after extended pleuropneumonectomy 284 days, and after decortication 315 days - significantly better than the prognosis in patients without surgical treatment or exploratory thoracotomy. Pleuropneumonectomy should only be considered in young patients with an epithelial cell-type tumour (possibly with adjuvant chemotherapy). Decortication seems nowadays to be the treatment of choice.

Does ND-YAG laser extend the indications for resection of pulmonary metastases?

Surgery forms part of a combined oncological concept in the management of pulmonary metastases. The following questions are relevant for its role: Does survival depend on the type of primary tumor? Are there any prognostic factors? What are the limits on radical resection? We analyzed retrospectively 657 patients who had undergone 759 resections of pulmonary metastases between 1973 and 1990. After conducting in vitro and in vivo experiments with the non-contact neodymium aluminum garnet (Nd-YAG) laser with a generating and delivery power of 10-120 W at the site of operation for 0.1-9.9 s, we have treated 65 patients by laser resection and/or vaporization since January 1990. Our preferred surgical approach was median or transverse thoracotomy. The 5-year survival of all resected patients was 30%, ranging from 21% (soft tissue sarcoma) to 60% (testicular carcinoma). Statistically significant differences in prognosis were seen related to the type of primary tumor, the disease-free interval, the caval or portal type of metastatic spread, the number of metastases and the potential degree of radical resection. The potential degree of conventional radical resections (wedge, anatomical sub-/segmental) was negatively influenced by the number of metastases (n > 9: 79% "radical" surgery = 38% 5-year survival). Laser treatment allowed parenchyma-preserving resection in cases of metastases of more than 0.5 cm in diameter, and vaporization in smaller ones. Resection with the intention of achieving complete remission was possible in up to 72 unilateral metastases. The complication rate was comparable to conventional resections.

HOPE technique enables Western blot analysis from paraffin-embedded tissues.

In contrast to the spectrum of biochemical analyses of fresh material, that of archived specimens is widely restricted. Fixation of specimens with formalin, the most commonly used fixative, usually prevents further molecular analysis, since it leads to degradation of nucleic acids and denaturation of the antigenic determinants of proteins. To overcome these problems, the Hepes-glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-fixation technique has been developed, which preserves nucleic acids and antigenic determinants of proteins, thus expanding the applicability of immunohistochemical methods. In this study, we investigated whether HOPE-fixed tissue can be analyzed by Western blotting. Furthermore, a comparison with conventionally fixed and frozen material was made. The specimens used were tumor-free and obtained from lobectomies for lung cancer. All four antibodies tested, i.e., antibodies specific for focal adhesion kinase, surfactant protein A, PI-3-kinase, and IKKalpha, worked well if used for immunoblotting of HOPE-fixed and frozen tissue. By contrast, these antibodies showed no or only very weak specific binding if formalin-fixed specimens were analyzed. Our findings show that HOPE fixation maintains the antigenicity of proteins better than formalin fixation. The possibility for performing Western blotting with archived paraffin-embedded specimens extends the options for diagnostic and scientific analyses of fixed tissues.

Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues.

We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment, permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degrees C) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.

[Reintervention in C-cell carcinoma]. / Reintervention beim C-Zell-Karzinom.

Cervical re-exploration in persistent medullary thyroid cancer usually fails to normalize serum calcitonin levels, which is the most sensitive criterion of tumour-free status (2 out of 21 patients in our re-exploration series). Positive lymph nodes - even at an early tumour stage - seem much more important (postoperative normal serum calcitonin: 86% in the occult tumour group, 71% in patients with palpable primary tumour and negative lymph nodes, as opposed to only 18% with a palpable cervical mass and positive lymph nodes). However, local re-exploration in case of persistent medullary thyroid cancer seems to offer a possible curative chance for the control of recurrence, especially after inadequate primary surgery. In cases without visible distant metastases a marked reduction in serum calcitonin level may be expected (21% of the preoperative level for stages N1 and N2 and 16% for stage N3 on average). In patients with elevated calcitonin levels after stimulation as sole indicator of persistent tumour the indication for reoperation should be handled cautiously. Thus, in 3 out of 5 patients with occult medullary thyroid cancer diagnosed only on the basis of venous sampling who were subjected to multiple cervical re-explorations, distant metastases were subsequently found during follow-up.

Surgery for congenital malformations of the lung.

In the course of a survey conducted in 59 hospitals performing thoracic surgery, 14 hospitals supplied data that could be used for the study. Out of 1347 anomalies diagnosed 1343 were surgically treated, with a 30-day mortality rate of 0.3% (5 patients). In a retrospective study over a period of 10 years (1978-1988) we identified 198 anomalies out of a total of 6350 thoracotomies; so our percentage grading of pulmonary anomalies is supported by the data of the above-mentioned survey according to which cystic pulmonary malformations such as inhibition malformations, excess malformation and lobar emphysema represent a majority with 72.2% (survey 83%). Congenital anomalies of lung formation occurred in 23% of the patients of the survey and in 15% of our own patients. Therapy consisted of parenchyma-saving surgery, i.e. enucleation (n = 87), segmental resection (n = 65) and lobectomy (n = 63) with bronchoplastic reconstruction; there was no 30-day mortality. Adenomatoid-cystic malformation, lymphangiectasis, congenital lobar emphysema and stenosis of the tracheobronchial tree are often an indication for immediate surgical treatment in neonates. Solitary cysts, bronchiectasis, sequestration of the lung, an AV-fistula present with symptoms mostly between the ages of 20-40 and therefore were surgically treated secondarily.