Assessment of a compounded synthetic adrenocorticotropic hormone product in 17 healthy dogs.

OBJECTIVES: Synthetic adrenocorticotropic hormone or tetracosactide is routinely used in the diagnosis of hypoadrenocorticism and frequently in the diagnosis and treatment of hyperadrenocorticism. There have been repeated shortages of tetracosactide in recent years in Australia. This study investigated the agreement of serum cortisol after a compounded tetracosactide (Bova Aus), compared to commercial tetracosactide (Synacthen®) in healthy dogs. METHODS: Prospective crossover study using 20 dogs. Ten dogs received 5 µg/kg Synacthen® on day 1 and 5 µg/kg compounded tetracosactide on Day 2. The other 10 dogs received the reverse order. Cortisol concentrations in each dog 1 h after injection were compared for agreement, which was defined as the limits of agreement of the Bland-Altman ratio to be within a range of 0.8-1.25. Passing-Bablok regression analysis examined for constant and proportional biases. RESULTS: Three dogs were excluded with post-stimulation serum cortisol concentrations markedly outside reference interval. For the remaining 17 dogs, Bland-Altman ratio analysis of cortisol concentration (tetracosactide/Synacthen®) at 1 h found virtually no constant bias (mean of ratios 1.01;95% CI 0.97-1.05) and 95% limits of agreement were 0.88 (95% CI 0.78-0.90) and 1.17 (95% CI 1.13-1.25). This met our criteria for agreement between cortisol concentrations. Bias of the Bland-Altman difference was 2.8 nmol/L (95% CI -7.2 to 12.8); 95% limits of agreement -35.2 nmol/L (95% CI -57.0 to -26.1) and 40.8 nmol/L (95% CI 31.7-62.6). Passing-Bablok regression analysis did not identify bias. CONCLUSION: In healthy dogs, cortisol concentrations were in agreement after compounded tetracosactide compared to commercial tetracosactide, Synacthen®.

A multivalent HIV-vaccine: development of a plasmid DNA for the expression of HIV envelope glycoproteins with hypervariable V3-loop domains.

The third hypervariable (V3) loop of the HIV-1 envelope glycoprotein gp120 plays an essential role in the process of viral entry. It contributes to the tropism, coreceptor usage and immune-escape of the virus. We generated a monovalent plasmid DNA and demonstrated the expression of HIV-1 clade B subtype NL4-3 gp120 and gp160 in comparison to a multivalent plasmid DNA encoding for a variety of V3-variants. In contrast to the membrane-anchored gp160, preliminary data demonstrate the monovariant gp120 is expressed in and presented on a human dendritic cell (DC) line, due to a HIVenv-specific re-stimulation of naïve T-cells detected by IFNgamma-ELISPOT assay.

[Neoadjuvant therapy in non-small cell lung cancer. Prognostic impact of "mediastinal downstaging"]. / Neoadjuvante Therapie des nichtkleinzelligen Lungenkarzinoms. Prognostische Relevanz des "mediastinalen Downstagings"

In the course of a prospective multicenter study, 40 (26 squamous cell and 14 adenocarcinomas) patients with stage IIIA and IIIB non-small cell lung cancer (NSCLC) were submitted to surgery after neoadjuvant radiochemotherapy. Pretherapeutic clinical lymph node status was compared to the lymph node involvement established in the resection specimens. Therapy-induced tumor regression was classified according to a three-step tumor regression grading system. In 29 patients (72.5%) a downward shift in lymph node involvement could be established,whereas in 27.5% ( n=11) pretherapeutic lymph node status was maintained. Of 26 patients with post-therapeutic N0 or N1 status, 21 revealed less than 10% vital tumor tissue in the resection specimens (regression grades IIb or III). Patients with post-therapeutic N0 or N1 lymph node status were found to have a survival benefit compared to patients with N2 lymph node involvement, though this difference was not statistically significant (p=0.27). On the other hand, tumor regression showed a significant correlation to the overall survival period (p=0.02). Thus, therapy-induced tumor regression grading seems to be a more precise method to predict the outcome of the disease.

Grading of tumor regression in non-small cell lung cancer : morphology and prognosis.

OBJECTIVE: Different types of multimodality therapy, including chemoradiotherapy and surgery, increasingly are being used for the treatment of patients with locally advanced non-small cell lung cancer (NSCLC; stages IIIA and IIIB). In this context, the applicability of a morphologic regression grading and its prognostic value were investigated. PATIENTS AND METHODS: In a multicenter phase II trial, 54 patients with locally advanced NSCLC received neoadjuvant bimodality treatment (ie, two cycles of ifosfamide, carboplatin, and etoposide, followed by twice-daily radiation up to 45 Gy with simultaneous administration of carboplatin and vindesine). Forty patients underwent resections. Using the corresponding resection specimens of the primary and regional lymph nodes, the following regression grading was established: grade I, no regression or only spontaneous tumor regression; grade II, morphologic evidence of therapy-induced tumor regression with at least 10% (grade IIa) or < 10% (grade IIb) vital tumor tissue; and grade III, complete tumor regression with no evidence of vital tumor tissue. Regression grading then was correlated with the survival time. RESULTS: Three tumors were classified as regression grade I, 10 were classified as regression grade IIa, 20 were classified as regression grade IIb, and 7 were classified as regression grade III. Patients with tumors of regression grades IIb or III showed significantly longer survival times than those with tumors of regression grades I or IIa (median survival time, 36 vs 14 months, respectively; 3-year survival rate, 52% vs 9%, respectively; p = 0.02). These survival times were also compared for patients who had undergone complete resection (median survival time, not reached vs 23 months, respectively; 3-year survival rate, 56% vs 11%, respectively; p = 0.03). The presurgical clinical response after patients had received neoadjuvant multimodality therapy had no predictive value in assessing the extent of therapy-induced tumor regression in the resection specimen. CONCLUSIONS: After neoadjuvant therapy of patients with NSCLC, the proposed tumor regression grading was of predictive value for long-term survival. Beyond the achievement of complete tumor resection (R0), a therapy-induced tumor regression of < 10% of vital tumor tissue is pivotal for superior long-term outcomes.

Ossification in atherosclerotic carotid arteries.

BACKGROUND: Heterotopic ossification as newly formed bone in extraosseous tissue is an uncommon finding in atherosclerotic lesions. The exact mechanisms and development of bone formation in regard to late stage calcified atherosclerosis still remains under debate. METHODS: We studied 400 autopsy carotid probes and 306 samples of atherosclerotic carotid endatherectomy. Radiographic analysis and classification of calcification was performed followed by light microscopy. In probes with detected ossifications further analysis using immunohistochemistry, scanning electron microscopy (SEM) and energy dispersive x-ray micro-analysis (EDX) including calcium mapping was performed. RESULTS: Ossification in atherosclerotic carotid arteries was a finding in only a minority of samples (5%) and occurred at sites of large calcific deposits. Histomorphology of bone formation equaled skeletal bone showing osteoblastic cells, osteocytes included in osteoid matrix, bone marrow and osteolytic giant multinucleated cells. Closely related to newly formed bone zones of neovascularization were found. Development of ossification seemed to occur in five stages (lipidous plaque, fibrous cellular plaque, fibrous acellular plaque, calcified plaque and osteogenesis). The environment of sites of ossification was characterized by a varying texture of extracellular fibrous matrix, foam cells, smooth muscle cells, fibroblasts and calcified deposits. CONCLUSIONS: Heterotopic ossifications of atherosclerotic plaques seem to be a specific differentiation of fibrous plaques. Components of atherosclerotic lesions like vascular wall cells, neovessels and matrix structures seem to be involved in the process of transformation to mature bone tissue.

[Changes in differentiation of small cell lung cancer after chemotherapy]. / Differenzierungswandel kleinzelliger Lungenkarzinome nach Chemotherapie.

We reexamined biopsy and resection specimens from seven patients with histologically established primary diagnosis of small-cell lung cancer (SCLC) which showed previously undetected adenocarcinomatous differentiation after chemotherapy. Adenocarcinoma was diagnosed in the same part of the lung as was SCLC before treatment in five cases and in contralateral location in two. Two tumors showed heterogeneous differentiation with small-cell and adenocarcinomatous components, and five had only adenocarcinoma. The interval between histological evidence of SCLC and adenocarcinoma was 3 months-5 years. From the specific findings in the respective patients we can offer two possible explanations for changing differentiation in SCLC after chemotherapy: (a) a primarily heterogeneously differentiated tumor with selection of adenocarcinomatous component under chemotherapy, and (b) an independently developed adenocarcinoma after successful chemotherapy of SCLC, as patients with treated SCLC are known to have an increased risk of non-SCLC. However, therapy-induced alteration of differentiation seems much more unlikely. These findings should be considered in diagnosis and therapy of SCLC to allow early and appropriate reaction to non-SCLC components.

[Synovial sarcoma of the pericardium]. / Synovialsarkom des Perikards.

A precordial tumor of the pericardium was radiologically diagnosed as the cause of an untypical clinical picture of heart disease in a 41-year-old soldier. As the patient had an increased asbestos exposure due to his profession, he was admitted to operation under the tentative diagnosis of a pericardial mesothelioma and the question of an occupational disease (BK 4105). Microscopic and immunohistochemical findings are compatible with the diagnosis of a synovial sarcoma of the pericardium. The present immunohistochemical marker spectrum allowed a reliable differentiation between synovial sarcoma and pericardial mesothelioma, which is more frequent than synovial sarcoma. The epithelioid component was determined using the following antibodies: MNF 116, CK 19, CK 7, EMA and Ber EP-4 were positive while Factor VIII, Calretinin, S100, Vimentin, CEA, CD 31, bcl-2 and HBA-71 were negative. The sarcomatous component was determined with antibodies to Vimentin, bcl-2 and HBA-71 which were positive, and to MNF 116, CK 19, CK 7, Factor VIII, Calretinin, S100, EMA, CEA, Ber EP-4 and CD 31 which were negative. Synovial sarcomas of the pericardium in the lower anterior mediastinum or the myocardium are exceedingly rare. A causal relationship between tumor formation and an increased asbestos exposure--similar to the epidemiologically based experiences with pericardial mesothelioma--is not likely. Primary extrapericardial synovial sarcoma could be excluded.

Internalization of human rhinovirus 14 into HeLa and ICAM-1-transfected BHK cells.

Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection.

Differential expression of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 in experimental glomerulonephritis.

We examined the relation between glomerular expression of chemokines from alpha-subfamily (macrophage inflammatory protein-2, MIP-2) and beta-subfamily (monocyte chemoattractant protein-1, MCP-1) and infiltration of neutrophils and monocytes in antibody mediated glomerulonephritis in rats. In the accelerated model of nephrotoxic nephritis (NTN), glomerular expression of MIP-2 and MCP-1 genes correlated with the sequential migration of neutrophil and monocyte influx, respectively. These relationships were investigated further in the heterologous phase of NTN by applying various treatments known to modulate the severity of injury. Pretreatment with bacterial lipopolysaccharide resulted in greater injury, MIP-2 expression increased 25- to 50-fold, and the glomerular neutrophil count increased two- to fourfold. Both MIP-2 mRNA levels and neutrophil infiltration were reduced by additional pretreatment with IL-6, IL-1 receptor antagonist, soluble IL-1 receptor or soluble TNF receptor (Spearman correlation coefficient r = 0.897, P < 0.005). In the heterologous phase of NTN, different pre-treatments only resulted in trivial changes in MCP-1 expression and monocyte infiltration. In conclusion, glomerular MIP-2 gene expression correlates with neutrophil infiltration both temporally during the evolution of nephritis, and when glomerular injury is modified by treatment. Glomerular MCP-1 gene expression correlates with monocyte influx. The data show chemokines of alpha- and beta-subfamilies co-operative to cause selective and sequential migration of different leukocyte subsets during development of antibody mediated glomerulonephritis.

Modulation of antibody-mediated glomerular injury in vivo by IL-1ra, soluble IL-1 receptor, and soluble TNF receptor.

The severity of glomerular injury in the heterologous phase of NTN is dependent on proinflammatory cytokines including TNF alpha and IL-1 beta, and can be enhanced by LPS. We have previously shown that passive immunization against IL-1 beta and TNF partially abrogated the LPS effect in this model. In the present work, we have assessed the effects on glomerular injury of blocking and binding of IL-1 to its receptor by rh IL-1 receptor antagonist (IL-1ra) and by neutralizing IL-1 and TNF with rm soluble IL-1 receptor type1 (sIL-1Rt1) and rh sTNF receptor (sTNFr p55), respectively. Pretreatment with either IL-1ra, sIL-1Rt1, or sTNFr partially abrogated the effects of LPS and reduced albumin excretion from 45 +/- 8, 66 +/- 9, and 101 +/- 17 mg/24 hr at 13 +/- 4 (P < 0.02), 14 +/- 4 (P < 0.001), and 21 +/- 7 mg/24 hr (P < 0.001), respectively. Similarly, these inhibitors reduced the prevalence of glomerular capillary thrombi and the intensity of glomerular neutrophil infiltration. Glomerular thrombosis was reduced from 18 +/- 3%, 28 +/- 5%, and 25 +/- 7% to 3 +/- 2% (P < 0.002), 6 +/- 2% (P < 0.001), and 3 +/- 2 (P < 0.001), respectively, and glomerular neutrophil infiltration was reduced from 46 +/- 3, 54 +/- 2, 59 +/- 8 to 19 +/- 2 (P < 0.001), 25 +/- 2 (P < 0.001), and 28 +/- 2 neutrophils/50 glomeruli in section, respectively. Coadministration of both soluble receptors of IL-1 and TNF caused a further decrease in glomerular injury. The protective effect was also noticed at four hours after induction of nephritis, and even when these inhibitors were administered after the LPS injection and at the same time of induction of nephritis. All three treatments reduced circulating TNF concentration (down to 20%, 34%, and 0%, respectively) but without detectable glomerular TNF gene expression. Glomerular IL-1 beta mRNA levels were also reduced by 41%, 53%, and 67%, respectively, when assessed by densitometric analysis of Northern blots. In contrast, the glomerular expression of IL-1ra was not affected by its exogenous administration but was mildly reduced by sIL-1Rt1 and sTNFr, which demonstrates the potential role for host derived IL-1ra as an endogenous negative feedback mediator in the glomerulus. These results confirm the direct involvement of IL-1 and TNF in LPS-enhanced hNTN and demonstrate the potency of these inhibitors in modulating injury even when administered after LPS and in time of induction of nephritis. They were more specific and effective than passive immunization with polyclonal antibodies, and this demonstrates their potential usefulness in the management of nephritis.

Antiviral effects of different CD4-immunoglobulin constructs against HIV-1 and SIV: immunological characterization, pharmacokinetic data and in vivo experiments.

The CD4 cell surface antigen belongs to the immunoglobulin superfamily and is the primary receptor for the human immunodeficiency virus 1 (HIV-1). The high affinity interaction between HIV-1 and CD4 is mediated by the viral envelope glycoprotein gp120. Recombinant soluble CD4 (rsCD4) has been shown in vitro to be an effective inhibitor of HIV-1 and HIV-2 propagation in lymphoid cells. A variety of antibody-like molecules were constructed, consisting of different parts of the extracellular domain of CD4 fused to immunoglobulin constant regions. The fusion proteins were expressed in mammalian cell lines and purified via affinity chromatography. The specificity and anti-viral effects of the different CD4-immunoglobulin constructs against HIV were analysed by different immunological tests, i.e., immunofluorescence, neutralisation and in vitro assays. In pharmacokinetic studies, differences were found in serum half-life between the four- and two-domain CD4 constructs in cynomolgus monkeys and between glycosylated and deglycosylated CD4-Fc constructs in rabbits. In two in vivo experiments using the four-domain CD4-Fc in SIV-infected macaques, no beneficial effects were observed.

Immunomodulatory activity of recombinant IL-1 receptor (IL-1-R) on models of experimental rheumatoid arthritis.

Given the role of IL-1 in inflammation and in autoimmune diseases, studies were designed to examine the ability of IL-1 receptor (IL-1-R) to suppress inflammation in a model of chronic degenerative joint disease of adjuvant arthritis (AA) in Lewis rats and to suppress the development of a systemic lupus erythematosus (SLE)-like disease in MRL/lpr mice. IL-1-R was able to prevent the onset of the AA and, even if therapy started after the establishment of AA, the cytokine receptor was still able to reduce the degree of chronic inflammation and arrested its progress. Treating MRL/lpr mice with IL-1-R resulted in a decrease in the amount of autoantibodies and inhibited joint inflammation. Even in the established disease IL-1-R could reduce rheumatoid factors (RF) and autoantibodies, and the signs of a polyarthritis were inhibited.

Immunoregulation of SLE-like disease by the IL-1 receptor: disease modifying activity on BDF1 hybrid mice and MRL autoimmune mice.

Due to the immunopharmacological profile of the recombinant IL-1 receptor (IL-1-R) and its potential to modulate biological activity in various inflammatory autoimmune disease models, we further elucidated its disease modifying activity on the development of a systemic lupus erythematosus (SLE)-like disease in BDF1 hybrid mice and in MRL/lpr autoimmune mice. Treatment of BDF1 mice with the IL-1-R during the induction phase resulted in a strong inhibition of the development of a glomerulonephritis, prolonged the survival time and improved the survival rate. Even a therapeutic effect was demonstrated when this receptor was given after the appearance of clinical symptoms. Treating MRL/lpr mice, which develop spontaneously a SLE-like disease, with the IL-1-R resulted in an inhibition of the developing glomerulonephritis and splenomegaly, in a reduction of swollen lymph nodes and in a decrease of autoantibody formation. Even in the established autoimmune disease of MRL/1 pr mice the IL-1-R reduced proteinuria, the levels of autoantibodies and also improved the survival rate.

[Gene technology in production of modern drugs]. / Gentechnologie bei der Herstellung moderner Arzneimittel.

In the last years gene technology has influenced dramatically medical and biological disciplines. With the help of molecular biology it is possible to produce a variety of proteins in alternative ways. Further, substances can be produced which exist in nature only in trace amounts, and therefore can not be produced with conventional methods. However, there is no other scientific discipline which is discussed so controversially in public like genetic engineering. The "new" biology comprises a multitude of disciplines which are difficult to understand for non experts. Unfortunately, gene technology has become the new symbol for "uncontrolled" technology development. In this review article the basic concepts of genetic engineering will be explained. Further, the production of some recombinant drugs, which are already used in practice, will be demonstrated. Finally, some safety aspects concerning about risk assessment are discussed.

Autoantibodies in HIV-infected hemophilia patients against different epitopes on CD4+ lymphocytes and recombinant CD4.

We studied 684 sera obtained from 20 hemophilia patients with AIDS/AIDS-related complex (ARC), 89 asymptomatic HIV+, 76 HIV- hemophilia patients and 151 healthy controls for antibodies against recombinant CD4 (rCD4). Twenty-two percent of AIDS/ARC patients, 10% of asymptomatic HIV+ patients, 17% of HIV-patients, and 1% of healthy controls had anti-rCD4 antibodies. Purified anti-rCD4 antibodies did not react with human CD4+ lymphocytes. This may explain why formation of anti-rCD4 antibodies correlated neither with the occurrence of autoantibodies against CD4+ lymphocytes nor with a decrease in CD4+ cell counts. Antibodies that were eluted from CD4+ lymphocytes after sequential adsorption and elution with separated CD8+ and CD4+ cells reacted with CD4+ lymphocytes of only some healthy individuals, suggesting diversity of CD4 expression.

Effect of continuous lumbar epidural anaesthesia during labour on fetal transcutaneous carbon dioxide.

The effect of epidural anaesthesia during labour on fetal transcutaneous carbon dioxide (tcPCO2) was observed on 27 fetuses. Our results show that in the course of epidural anaesthesia there is an increase in fetal tcPCO2. We can see a slight increase even before administering the test dose, while preparatory measures are undertaken for the epidural anaesthesia. After administering the test dose and after giving the main dose there is a further increase in fetal tcPCO2, which continues for up to 30 min after the main dose has been given. The results suggest that it is important to consider any pathological conditions in the fetus, so as to avoid the possibility of additionally endangering the fetus during epidural anaesthesia.

Molecular biology of proteins involved in blood coagulation.

Normal hemostasis in humans requires the interaction of a large number of plasma glycoproteins with blood platelets and vascular endothelial cells. Many of the plasma glycoproteins which participate in blood coagulation are zymogens of enzymes that interact in a stepwise manner in a series of reactions. In the last years most of these glycoproteins have been purified from human plasma by standard techniques. Some of them are used as therapeutics for restoring coagulation disorders. The knowledge about the plasma proteins involved in blood coagulation was greatly increased after cloning and sequencing of the respective complementary DNAs. Furthermore, recombinant DNA technology is used for the alternative production of several coagulation factors. It is the aim of this article to give an overview about the molecular biology of the enzymes and cofactors involved in blood coagulation.

[Magnetoencephalography--current trends]. / Magnetenzephalographie--Gegenwärtige Trends.

The review deals with the method of magnet-encephalography. The physical principles and the most important technical conditions to put into practice this method are mentioned. Comparing with the electroencephalography this method shows various advantages, what leads to its intensive use for the localisation of components of evoked potentials, for the magnet-encephalographic record of the brain activity of the human fetus, and for the localisation of epileptic foci in therapy resistant epileptics.

The influence of oxygen administration to the mother during labor on the fetal transcutaneously measured carbon-dioxide partial pressure.

The aim of this study was to plot the course of the transcutaneously measured PCO2 (tcPCO2) in the fetus during oxygenation of the mother. In our examination 35 parturients with a suspicious or pathologic CTG were given pure oxygen for 10 minutes at a flow speed of 10 l/min. The fetal tcPCO2 was measured with a TCM 3 measuring device from Radiometer. The measuring temperature was 41 degrees C. The fetal tcPCO2 was 67.2 +/- 3.9 mmHg before the O2 application, during the O2 application it was 67.3 +/- 14.1 mmHg and for the period after the O2 application we found an average measurement of 66.7 +/- 13.9 mmHg. Further we investigated whether, depending on the original levels of the fetal tcPCO2 an O2 application to the mother had a measurable effect on the fetal tcPCO2 levels. The average levels of the tcPCO2 in the fetuses with pathological original levels of greater than or equal to 60 mmHg or with normal levels of less than 60 mmHg did not show any significant differences before, during or after the O2 application. Our own results and reports given in the literature about an increase in the fetal O2 partial pressure during maternal oxygenation lead to the conclusion that in cases with fetal hypoxia, the O2 application to the mother--in addition to other measures for intrauterine reanimation or speedy termination of labor--could be of advantage.