Bone marrow Th2 cytokine expression as predictor for relapse in childhood acute lymphoblastic leukemia (ALL).

The immunological environment of leukemic blasts in the bone marrow might play a decisive role in determining an individual's risk for relapse. In order to identify potential predictors of relapse and to elucidate the mechanisms of immune control of leukemic blasts we examined the expression of cytokines, costimulatory molecules and members of the TNF family in leukemic marrow samples in a prospective study. Samples from 49 consecutive pediatric patients with B cell precursor acute lymphocytic leukemia (BCP ALL) were analyzed by semiquantitative RT-PCR. We identified interleukin (IL)-10 expression as a significant adverse prognostic indicator in childhood BCP-ALL. The event free survival (EFS) of patients expressing IL-10 mRNA in high quantity was significantly lower compared with patients expressing low IL-10 mRNA. Taqman RT-PCR of sorted cell populations showed that IL-10 mRNA was synthetized almost exclusively by NK or T cells. In addition, we found an increased expression of IL-1, IL-4, CD86 and VEGF mRNA in patients with late relapses. Possibly, ALL cells mediate a Th2 shift through increased expression of CD86 and thereby influence the individual relapse risk. These findings emphasize the role of the immune system for the outcome of childhood ALL.

[Will the effectiveness of the immunization of chicks with live Salmonella vaccines be affected by maternal antibodies?]. / Wird die Wirksamkeit der Immunisierung von Küken mit Salmonella-Lebendimpfstoffen durch maternale Antikörper beeinflusst?

In the progeny of breeder birds which had been vaccinated with live Salmonella Typhimurium and inactivated Salmonella Enteritidis vaccines, the caecal and systemic colonisation by a live Salmonella Enteritidis and a live Salmonella Typhimurium vaccine was studied. The efficacy of the oral immunisation of chicks from vaccinated and non-vaccinated breeders with a live Salmonella Enteritidis vaccine on day 1 of age was studied by an experimental challenge with Salmonella Enteritidis on day 30 of age. Antibody production of isotypes IgG, IgA and IgM was determined in sera and jejunum of the birds. Vaccination of parent birds resulted in an increase of the antibody concentration in sera and jejunum of the chicks. Own antibody production after administration of the live Salmonella vaccine to the day-old chicks was not detected until day 21 of life. Compared to controls, the number of vaccine organisms in the caeca of the progeny of vaccinated breeder birds was reduced by 0.5-1.5 log10 units. The reduction of the Salmonella Enteritidis vaccine was more pronounced than that of the Salmonella Typhimurium vaccine. However, the reduced colonisation by the live Salmonella vaccine strain did not impair the efficacy of the immunisation of the chicks. To ensure efficacy of the active oral immunisation of chicks from vaccinated parent birds with attenuated live Salmonella vaccines also in case where amounts of maternally transferred antibodies are even higher, it should be guaranteed that chicks take in via drinking water the recommended dose of the vaccine strain. In this connection, factors like the low intake of drinking water by very young chicks, the concentration of the vaccine organisms in the water and the survival of the vaccine should also be considered.

[Necessity of double testing of meat juice samples from pigs using ELISA for the determination of Salmonella status in pig farms]. / Uber den Nutzen von Doppelbestimmungen bei Fleischsaftuntersuchungen mittels ELISA für die Beurteilung des Salmonellenstatus von Schweinebeständen.

According to the test protocol of the "meat juice ELISA" for detection of salmonella antibodies in pigs, all meat juice samples and serum controls are to be tested in duplicate. Results from routine investigations of repeatedly double tested meat juice and serum samples have been used to analyze the effect of double testing versus single testing with regard to the reliability of the final result. In case of an individual animal, testing of samples in duplicate increases the reliability of the results significantly, especially, if samples are retested at different occasions. In contrast, such a difference between mono and double testing of samples is not of importance when a group of animals is tested in order to determine the mean antibody rate in a herd. Here, results from double testing practically do not contribute to a higher reliability of the final result. This observation provides the possibility to reduce the costs for investigation programmes drastically.

[Subepidermal calcified nodule (calculus) of the eyelid]. / Subepidermaler kalzifizierter Knoten (Kalkulus) des Lides.

A 9-year-old boy presented with a painless hard tumor in the right lower lid. It had grown slowly since 12 months. Three months before presentation cryotherapy was administered by a dermatologist because of a presumed wart. Both eyes were normal. Clinical differential diagnosis included a keratoakanthoma and a wart. Excisional biopsy was performed. Histology revealed granular deposits which stained positive with hematoxylin-eosin and von Kossa stain. Though a secondary (dystrophic) calcinosis of the lid following cryotherapy could not be ruled out a diagnosis of an idiopathic subepidermal calcified nodule (calculus) of the lid was established.

Effects of mycophenolate mofetil on nasal mucosal tolerance induction.

PURPOSE: The authors investigated mucosal tolerance therapy as a treatment for autoimmune conditions, including uveitis. Although nasal antigen administration was unable to suppress the disease when given to primed animals, previous studies of experimental autoimmune uveoretinitis (EAU) have shown that nasal antigen administration can maintain disease suppression when combined with oral cyclosporin A. This study aimed to determine whether mucosal tolerance can be induced when EAU is suppressed with mycophenolate Mofetil (MM) and whether tolerance can be maintained when immunosuppression with MM is stopped. METHODS: Lewis rats were immunized with retinal extract, and then they received either oral MM 7 to 20 days after immunization or retinal extract intranasally in combination with oral MM on days 7 to 20. Thereafter, weekly nasal administration of the antigen was given until the termination of the experiment at day 38. One group of control animals received the drug vehicle orally and phosphate-buffered saline intranasally. Clinical and histologic changes were assessed along with changes in immune status including delayed-type hypersensitivity, antibody responses to retinal antigens, and flow cytometric phenotyping of infiltrating ocular leukocytes. RESULTS: EAU was delayed, but not prevented, by a short-term course of MM (7-20 days after immunization). Tolerance to the retinal extract could not be induced during MM treatment by nasal retinal extract administration. Despite the delay in onset of EAU in MM and in MM- and nasal antigen-treated animals, profound target organ damage occurred as seen in untreated controls with EAU. However, fluoroscein-activated cell sorter analysis of retinal leukocytic infiltrate indicated that there was a reduced macrophage recruitment at all time points, whereas lymphocyte infiltration was reduced in proportion to the overall reduction in leukocyte infiltration during therapy. CONCLUSIONS: Nasal retinal antigen administration does not induce tolerance or maintain disease suppression when combined with MM therapy during the effector stage of the (auto)immune response. MM therapy delays disease onset, but target organ damage occurs when therapy is stopped, despite a marked inhibition of macrophagemonocyte infiltration into the chorioretina.

Cataract surgery at the end of the 19th century at Tübingen.

Cataract extraction with a Graefe knife incision was the most important development in cataract surgery during the 19th century. To determine the indications, visual outcome, complications, and problems of cataract surgery performed a century ago, we reviewed patient records from the year 1895. With use of Graefe's technique of cataract extraction, the early postoperative visual acuity was 20/200 or better in 63% and 20/40 or better in 5%. A secondary cataract developed in about 30% of eyes. A flat anterior chamber persisted for more than two days in about 20% of eyes. Astigmatism was not regularly measured, but was markedly increased after surgery. Surgery for secondary cataract was performed in only 20 eyes. There was a complication rate of nearly 50% in secondary cataracts. These results demonstrate some of the major problems of cataract surgery 100 years ago: secondary cataract, insufficient wound closure, high astigmatism, and aphakia as a refractive problem.

Phenotypic analysis of retinal leukocyte infiltration during combined cyclosporin A and nasal antigen administration of retinal antigens: delay and inhibition of macrophage and granulocyte infiltration.

Nasal antigen administration successfully suppresses a model of organ-specific autoimmune disease, experimental autoimmune uveoretinitis (EAU), when administered prior to immunisation. We have previously shown that nasal antigen therapy for active disease or in primed, sensitised animals does not reliably or consistently suppress histological disease. However, when nasal antigen administration is combined with cyclosporin A (CsA) therapy, rod outer segment destruction (target organ) is reduced despite the presence of clinical and histological leukocytic infiltration of the eye. In this study, two colour flow cytometric phenotypic analysis of retinal and choroidal leukocytic infiltration of animals treated with either CsA alone, combined therapy with CsA and inhalational tolerance therapy with retinal antigens or sham treated controls was performed. There was no clinical difference between the two treated groups. Flow cytometric phenotypic analysis was performed in all groups at both maximal clinical disease and during resolution of clinical signs. Although the cell number within the infiltrate was reduced in combined treated group, CD4+ IL-2R+ T cells were still present in large numbers, in contrast to the markedly reduced numbers of ED7+ (macrophages/granulocytes) cells infiltrating during height of disease. In the CsA-nasal antigen treated group, when clinical inflammation had subsided, an increase in both macrophages and granulocyte numbers in the chorioretina was observed. The cell numbers were always less than CsA-only treated animals. Despite the late cellular influx of monocytes/macrophages, rod outer segment (ROS) integrity as determined histologically, was maintained. Nasal antigen administration of retinal antigens in CsA-only treated animals (combined therapy group) protects against target organ damage without inhibiting activated T cell traffic to the eye. These results suggest that recruitment of macrophages to the target tissue is central to autoimmune target organ damage, the mechanisms of which are discussed.

Nasal administration of retinal antigens maintains immunosuppression of uveoretinitis in cyclosporin-A-treated Lewis rats: future treatment of endogenous posterior uveoretinitis?

PURPOSE: Current treatment of autoimmune endogenous posterior uveoretinitis (EPU) is limited by drug toxicity, unpredictable relapses on dose reduction and resistance to therapy. Administration of autoantigens via gastrointestinal or respiratory mucosa prior to antigen exposure induces immune hyporesponsiveness (mucosal tolerance) to further antigen sensitisation. In this study we assessed whether mucosal tolerance induction was possible after immunisation with retinal antigens in experimental autoimmune uveoretinitis (EAU) in animals that were short-term immunosuppressed with cyclosporin A (CsA) to determine whether mucosal administration of retinal antigens can maintain immunosuppression in sensitised and immunosuppressed individuals. METHODS: Female Lewis rats were immunised with retinal extract (RE) and then treated as follows. Group 1 received no specific therapy and served as control; group 2 were fed CsA from day 7 to day 20 post-immunisation; group 3 received inhalational tolerance therapy with RE in addition to CsA; tolerance therapy was continued after day 20 when CsA was stopped. Experiments varying the timing and dosage of both tolerising and immunising antigen were also performed, the details of which are described. Incidence, day of onset and clinical activity were recorded and histopathological assessment of intraocular inflammation, in particular the extent of autoimmune target-organ damage, was graded semiquantitatively. RESULTS: Compared with controls and group 2, group 3 showed both a marked delay in disease onset and a reduction in disease severity. This effect was both dose and dose-timing dependent. Tissue damage assessed in terms of preservation of rod outer segments was significantly less in group 3. CONCLUSIONS: The success of combination therapy, clinically, remains unknown at present but these results support continuing present clinical trials of mucosal tolerance therapy and in particular have future implications for either maintaining or inducing immunosuppression in autoimmune diseases in combination with present immunosuppressive therapies.

[Diode laser cyclophotocoagulation in treatment of therapy refractory glaucoma]. / Diodenlaser-Zyklophotokoagulation in der Behandlung therapierefraktärer Glaukome.

BACKGROUND: It has been suggested that Nd:YAG laser cyclophotocoagulation significantly lowers the intraocular pressure in refractory glaucoma. However, only limited information is currently available concerning the cyclodestructive effects of diode laser cyclophotocoagulation. PATIENTS AND METHODS: Twenty eyes of 17 patients had undergone diode laser cyclophotocoagulation if previous glaucoma surgery including cyclocryocoagulation was unsuccessful or the patients were blind. Nine eyes had secondary glaucoma, 5 neovascular glaucoma, 5 congenital glaucoma, and 1 eye chronic open angle glaucoma. Treatment was applied in two quadrants using a 810 nm diode laser (600 qm diameter quartz fiber, 2 sec, 1.75-3 W, 6 to 10 pulses). Seven eyes required retreatment. RESULTS: In 19 eyes the baseline IOP was 32.1 +/- 10.1 mm Hg and at the patient's last examination 21.0 +/- 8.4 mm Hg. Median follow up was 11 months (3 to 19 months). In 4 of 5 eyes with a visual acuity of less than 1/50 IOP was decreased under 30 mm Hg, while 9 of 14 eyes with a visual acuity of at least 1/50 IOP revealed a decrease of IOP under 20 mm Hg, resulting in a success rate of 68%. Complications included acute severe anterior uveitis in one eye and bulbus hypotonia in another for several months. After three weeks, one eye was enucleated because of persisting high IOP and pain. Histopathologically, coagulation necrosis of the ciliary body stroma and separation of epithelial layers were observed. CONCLUSIONS: This study suggests that contact diode laser cyclophotocoagulation is an effective method and a valid alternative to cyclocryocoagulation in the treatment of refractory glaucoma including congenital glaucoma.

Oculomucocutaneous changes as paraneoplastic syndromes.

Bullous dermatoses such as erythema exsudativum multiform major (EEMM) and bullous pemphigoid can lead to severe ocular involvement. In rare cases, both diseases develop as paraneoplastic syndromes. The immunopathologic mechanisms are discussed. A 69-year-old woman with non-Hodgkin's lymphoma (NHL) of grade IIIb developed EEMM while under systemic treatment with Fluconazole, Ofloxacin, and/or a combination of sulfamethoxazole and trimethoprim after polychemotherapy. In the eye, conjunctival necrosis with sicca syndrome led to Staphylococcus aureus-induced corneal superinfection, perforation, and consecutive keratoplasty à chaud. The patient died 6 weeks after the first presentation. A 44-year-old man with NHL of grade IVa after polychemotherapy developed a bullous pemphigoid affecting the skin, mucous membranes, and both eyes while under systemic treatment with sulfamethoxazole and trimethoprim. Although the underlying malignancy responded well to chemotherapy, the ocular manifestations of the paraneoplastic systemic syndrome slowed down only on treatment with cyclosporin A but not following therapy with azathioprine and cyclophosphamide. Therapy could not stop cicatrization and keratinization of the conjunctiva and cornea. An occult malignancy should be excluded in acute and chronic oculomucocutaneous syndromes. The prognosis for the eye seems to be poor because of the rapid course and the ineffectiveness of therapy as demonstrated in the present cases.

[The specificity of antibodies reacting with Salmonella antigens in ELISA]. / Untersuchung zur Spezifität der im ELISA mit Salmonellaantigen reagierenden Antikörper.

Among cattle sera from herds without a history of salmonellosis and without vaccination against salmonellas, we sometimes found samples which reacted positively in the ELISA with salmonella antigen. Contrary to antibodies caused by a proved salmonella infection, these antibodies could be eliminated by a whole-cell lysate of E. coli. In the colostra of cows which had received a salmonella live vaccine orally in the first days of life but were raised and kept without known exposition to salmonellas, we also found antibodies which reacted in the salmonella ELISA. They could not be eliminated by E. coli lysate.

Course of infection and humoral immune reaction in calves infected orally with different Salmonella serovars.

5 groups of five calves each were orally infected with the salmonella serovar S. agona, S. dublin, S. enteritidis, S. infantis or S. typhimurium. The course of infection and the humoral immune response against 5 antigen preparations of the homologous and the 4 heterologous salmonella serovars were monitored in each group. Antibodies against two different protein antigens and against two differently prepared LPS antigens were determined by ELISA and the level of H-agglutinin was assessed by micro agglutination test. All 5 serovars proved to be virulent for calves. The specificity of antibodies produced following the infection are not always consistent with the Kauffmann-White scheme. There was a relation of age and strain for infection on immune response, whereas the severity of infection did no correlate with an increase of antibody production. The serological tests used were good for investigation of a defined host-parasite-interaction but not satisfactory for serovar specific diagnosis of salmonella infection in cattle.

[Diagnostic potential of biochemical and cytological parameters in bronchoalveolar lavage fluid in healthy calves and calves with pneumonia]. / Diagnostische Aussagefähigkeit biochemischer und zytologischer Parameter in der Lungenspülflüssigkeit gesunder und pneumoniekranker Kälber.

Bronchoalveolar lavage fluid (LF) was obtained from cranial as well as from caudal regions of the bovine lung in healthy and pneumonic calves. LF was investigated biochemically, cytologically, and bacteriologically. In healthy lungs free of pneumonic lesions, no differences due to the different parts of the lung were established. An increase in both protein content and number of cells and a marked influx of neutrophils were found to be the most important alterations caused by pneumonic lesions. The diagnostic potential of lactate dehydrogenase, alkaline phosphatase and the size of alveolar macrophages in LF was validated.

[Induction of gamma-glutamyltransferase in cultured brain cells]. / Induktion der gamma-Glutamyltransferase bei Hirnzellen in Kultur.

gamma-Glutamyltransferase is a membrane-bound enzyme widely distributed in animal tissues. This enzyme is involved in glutathione metabolism, but its exact biological function is still an open question. In rat brain cells in culture a depletion of L-cystine and L-glutamine or the addition of diethyl maleate to the culture medium leads to a decrease of intracellular glutathione concentration and to an increase of the specific activity of gamma-glutamyltransferase. The induction of the enzyme is inhibited by the addition of cycloheximide or actinomycin D to the culture medium.

[Induction of alkaline phosphatase by retinoic acid]. / Induktion der alkalischen Phosphatase durch Retinsäure.

Treatment of mammalian cells in culture with retinoic acid causes a time- and concentration-dependent increase of the specific activity of alkaline phosphatase. The increase reaches a factor of 15 and more and begins at a concentration of 10(-8)M retinoic acid. The induction is inhibited by cycloheximide or actinomycin D. The same isoenzyme of alkaline phosphatase is expressed in control and in retinoic acid-treated cells as demonstrated by the inhibitions by amino acids and peptides. The enzyme induction occurs in rat heart, skeletal muscle, brain, lung cells and HeLa cells. No induction was found in two lines of human melanoma cells. After treatment of cells with tunicamycin, the induction of alkaline phosphatase is detectable only in the homogenate and no longer detectable by histochemical methods. This shows that the glycosylation of the protein is an important step in the insertion of this enzyme into the plasma membrane.

[Studies of carbohydrate absorption in clinically healthy and diarrheal calves]. / Untersuchungen zur Absorption von Kohlenhydraten bei klinisch gesunden und an Durchfall erkrankten Kälbern.

Reported in this paper are results obtained from functional and morphological studies into absorption of carbohydrates by clinically intact and diarrhea calves. In that context, the xylose-stress test was used for functional determination of enteral absorption. Diarrhoea, caused in the calves by several pathogens, such as rotavirus, Escherichia coli, and Salmonella dublin, led to significant impairment of carbohydrate absorption in the gastro-intestinal canal. However, glucose absorption was not even fully stopped by lasting severe diarrhoea over several days. Glucose-containing special diet drinks, therefore, may help, at least partially, stabilising the glucose metabolism of calves treated for diarrhoea. A comparison between functional and morphological tests for enteral absorption seemed to suggest that histological findings should not be overestimated regarding information obtainable from them on impaired functionality of intestinal mucosa.

How to collect blood for the measurement of HGH, LH, FSH, cyclic AMP, folic acid, cortisol and testosterone.

In order to estimate peptide hormones, steroid hormones, folic acid and cyclic AMP in the same blood sample we investigated the influences of different anticoagulants on the measurement. It could be demonstrated that blood collected in EDTA-coated tubes can be for the measurement of each of the mentioned constituents.