A multifunctional test mixture for chiral cyclodextrin GC columns.

Gas chromatography (GC) is a powerful tool in the separation science of chiral analytes. The development of a new chiral test mixture for cyclodextrin (CD) coated GC columns allows comparing, choosing and identifying most suitable columns for a given separation challenge. This test mixture contains 12 enantiomer pairs of a broad range of functional groups and it is the first mixture suitable for all types of modified cyclodextrin capillary columns. Column changes can be observed and system performance can be monitored by means of this test mixture, which is therefore a base for reliable results. Furthermore, this publication is thought to be a start-up aid for chiral GC.

Differential effects of TNF and LTalpha in the host defense against M. bovis BCG.

Signaling via TNF receptor type 1 (TNFR1) was shown to be crucial in host defense against the intracellular pathogens L. monocytogenes, M. tuberculosis and M. bovis. To investigate the function of TNF and LTalpha in host defense against M. bovis, mice double deficient for TNF and LTalpha (TNF / LTalpha (- / -)), TNF / LTalpha (- / -) mice complemented with a murine LTalpha transgene (TNF(- / -)) and LTalpha (- / -) mice were infected with BCG and the ensuing pathology was investigated. Control mice showed a normal host defense with early clearance of bacteria. The granulomatous reaction in the liver was accompanied by recruitment of activated macrophages characterized by their acid phosphatase positivity and differentiation into epithelioid cells as well as a coordinated expression of proinflammatory transcripts. In contrast, TNF / LTalpha (- / -) mice showed no comparable recruitment of activated macrophages in the liver. Furthermore, these mice showed extensive necrotic pulmonary lesions with massive growth of acid fast bacilli. Reintroduction of LTalpha as a transgene into TNF / LTalpha (- / -) mice prolonged survival but did not restore resistance to BCG. This, at least partially protective role of LTalpha was further supported by data demonstrating that LTalpha -deficient mice as well were susceptible to BCG infection. In contrast to the deleterious effect of TNF / LTalpha deficiency in BCG infection, BCG-infected TNF / LTalpha (- / -) mice were tolerant to LPS-induced shock. These results demonstrate that TNF as well as LTalpha are involved in murine host defense against BCG and that absence of TNF / LTalpha protects BCG-infected mice from LPS mediated shock.

Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein.

A CD4-independent version of the X4 human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope (Env) protein, termed 8x, mediates infection of CD4-negative, CXCR4-positive cells, binds directly to CXCR4 in the absence of CD4 due to constitutive exposure of a conserved coreceptor binding site in the gp120 subunit, and is more sensitive to antibody-mediated neutralization. To study the relationships between CD4 independence, neutralization sensitivity, and exposure of CD4-induced epitopes associated with the coreceptor binding site, we generated a large panel of Env mutants and chimeras between 8x and its CD4-dependent parent, HXBc2. We found that a frameshift mutation just proximal to the gp41 cytoplasmic domain in 8x Env was necessary but not sufficient for CD4 independence and led to increased exposure of the coreceptor binding site. In the presence of this altered cytoplasmic domain, single amino acid changes in either the 8x V3 (V320I) or V4/C4 (N386K) regions imparted CD4 independence, with other changes playing a modulatory role. The N386K mutation resulted in loss of an N-linked glycosylation site, but additional mutagenesis showed that it was the presence of a lysine rather than loss of the glycosylation site that contributed to CD4 independence. However, loss of the glycosylation site alone was sufficient to render Env neutralization sensitive, providing additional evidence that carbohydrate structures shield important neutralization determinants. Exposure of the CD4-induced epitope recognized by monoclonal antibody 17b and which overlaps the coreceptor binding site was highly sensitive to an R298K mutation at the base of the V3 loop and was often but not always associated with CD4 independence. Finally, while not all neutralization-sensitive Envs were CD4 independent, all CD4-independent Envs exhibited enhanced sensitivity to neutralization by HIV-1-positive human sera, indicating that the humoral immune response can exert strong selective pressure against the CD4-independent phenotype in vivo. Whether this can be used to advantage in designing more effective immunogens remains to be seen.

The highly conserved C-terminal dileucine motif in the cytosolic domain of the human immunodeficiency virus type 1 envelope glycoprotein is critical for its association with the AP-1 clathrin adaptor [correction of adapter].

Short amino acid sequences in the cytosolic domains of transmembrane proteins are recognized by specialized adaptor [corrected] proteins which are part of coated vesicles utilized to transport membrane proteins between the trans-Golgi network (TGN) and the plasma membrane (forward and backward). Previously, we and others reported that the membrane-proximal tyrosine residues Y712 (human immunodeficiency virus [HIV]) and Y721 (simian immunodeficiency virus [SIV]) in the envelope glycoprotein (Env) of the primate lentiviruses are crucial for the association of Env with clathrin-associated adaptor [corrected] complex AP-2. The same tyrosine-based endocytosis motifs in the cytosolic domains (EnvCD) of transmembrane gp41 of HIV type 1 (HIV-1) and SIV, respectively, were also shown to modulate the interaction with TGN- and endosome-based clathrin-associated complex AP-1. Our findings suggested that EnvCD binding to AP-1, unlike the association of EnvCD with AP-2, is dependent largely on residues other than Y712 and Y721. Here, we tested if motifs downstream of Y712 affect HIV-1 EnvCD-AP-1 binding and Env trafficking. Mutational analysis revealed that the C-terminal leucine-based motif in Env was crucial for the recruitment of AP-1 in vitro and in Env-expressing cells. In addition to affecting Env-AP-1 association, mutations at the C terminus of Env also altered the subcellular localization of Env, suggesting that proper post-Golgi routing of Env depends on its recruitment of AP-1. Finally, the C-terminal dileucine was shown to assist the membrane-proximal Y712 motif in restricting the cell surface expression of Env.

Long-term results in patients treated with posterior instrumentation and fusion for degenerative scoliosis of the lumbar spine.

The authors report a homogeneously investigated and surgically treated series of 40 patients with degenerative scoliosis of the lumbar spine. The series included 22 females and 18 males with a mean age of 62.8 years. The clinical presentation, the diagnostic work-up, the indication for surgery, the surgical techniques and results are reported. Final evaluation was possible in 30 patients at a mean period of observation of 59.5 months. Following a very precise diagnostic and therapeutic protocol excellent, good and satisfactory surgical results were obtained in 13 (43.3%), 16 (53.3%) and 1 (3.3%) patients, respectively. While scoliosis was converted from a mean preoperative Cobb angle of 18.7 degrees to 7.6 degrees mean pre-operative lumbar lordosis was slightly augmented from 37 degrees to 41.5 degrees. The results suggest that maintainance or correction of lumbar lordosis is more important than the conversion of the scoliotic deformity which is probably treated sufficiently by partial correction and stabilization. Observation over time indicates that the degenerative cascade evolves despite internal fixation and fusion in the majority of the patients until a stable state is reached. This stable state is probably rather the result of ankylosis of the facet joints than the effect of posterolateral fusion.

A membrane-proximal tyrosine-based signal mediates internalization of the HIV-1 envelope glycoprotein via interaction with the AP-2 clathrin adaptor.

The envelope glycoprotein (Env) of human immunodeficiency virus, type 1 (HIV-1) undergoes rapid internalization after its transport to the cell surface. Env internalization is dependent upon information contained within the cytosolic domain of the protein. Here, we report that the cytosolic domain of Env binds specifically to the medium chain, mu 2, of the clathrin-associated protein complex AP-2, as well as to the complete AP-2 complex. The Env cytosolic domain contains two highly conserved tyrosine-based motifs (Y712SPL and Y768HRL), both of which are capable of binding to mu 2 when presented as short peptides. However, only the membrane-proximal motif Y712SPL binds to mu 2 and is required for internalization in the context of the whole cytosolic domain of Env. A glycine residue (Gly711) adjacent to the Y712SPL motif is also important for binding to mu 2/AP-2 and internalization. These observations suggest that the accessibility of the membrane-proximal GY712SPL to mu 2/AP-2 determines its function as a signal for recruitment of HIV-1 Env into clathrin-coated pits and its ensuing internalization.

Mononuclear blood cell sulfidoleukotriene generation in the presence of interleukin-3 and whole blood histamine release in honey bee and yellow jacket venom allergy.

Cross-linking IgE on basophils is known to cause both sulfidoleukotriene (sLT) generation and histamine release. We recently developed an ELISA to determine sulfidoleukotriene generation by blood mononuclear cells which employs pretreatment with IL-3 to enhance leukotriene generation (cellular antigen stimulation test, CAST). Here, we compared the CAST and whole blood histamine release in response to honey bee/yellow jacket venom (BV/YJV) in 23 patients clinically suspected of type-I allergy to these venoms. Of these, 17 were diagnosed as "definitive venom allergics," defined by a positive skin test at 100 ng/ml of venom or less. The six in whom such skin reactivity was absent were labelled "suspected venom allergics." Both venoms stimulated sulfidoleukotriene generation and histamine release also from control individuals (n = 10). In patients, insect venoms generally stimulated histamine release and sulfidoleukotriene generation in excess of the mean + 3 SD of values obtained with control individuals. However, about half of the patients reacted predominantly with either histamine release or sulfidoleukotriene generation. No overall correlation was found between threshold doses necessary to stimulate sulfidoleukotriene generation (ThsLT) and histamine release (ThHist). (Linear correlation coefficients between ThsLT and ThHist were -0.02 for honey bee venom and 0.13 for yellow jacket, n = 23). Both findings are in contrast to the concept that these responses occur in parallel. From results with "definitive venom allergics," CAST sensitivity was calculated as 100% for honey bee venom and 83% for yellow jacket, and that of the histamine release assay as 62.5% for honey bee venom and 50% for yellow jacket. Specificity of the CAST was calculated as 77% for honey bee venom and 100% for yellow jacket, and that of the histamine release assay as 44% for honey bee venom and 60% for yellow jacket. Thus, CAST results are closer to skin test results than to those of the whole blood histamine release assay.

[Basophil degranulation test (BDT) as a parameter of hyposensitization with Hymenoptera venoms]. / Der Basophilen-Degranulationstest (BDT) als Verlaufsparameter einer Hyposensibilisierung mit Hymenopterengiften.

In a population of 24 insect sting allergy patients undergoing venom immunotherapy the basophil degranulation test (BDT) in the patient sera ("unwashed" BDT) and with washed leukocytes ("washed" BDT) after incubation with bee and wasp (yellow jacket) venom was performed before and during treatment. Venom specific IgE and IgG antibodies, detected by means of RAST, were also monitored. The "unwashed" BDT usually became negative within 6-9 months of beginning immunotherapy, whereas the IgE-RAST was still clearly positive. This was attributed to the blocking influence of the venom specific IgG antibodies induced by the venom therapy. In fact, at this time the BDT with "washed" blood leukocytes, i.e. after elimination of the serum antibodies, was generally still positive. Only during further immunotherapy did cellular sensitization in the "washed" BDT gradually disappear, whereas the IgE-RAST usually turned out weakly positive. A third of the patients showed simultaneously negative results of "unwashed" and "washed" BDT, independently of venom specific IgE and IgG levels. These findings suggest a specific reactivity change of the blood basophils (cellular desensitization) induced by the immunotherapy. The BDT can be used as an immunological parameter for IgG-monitoring of the course of venom immunotherapy and--in addition to skin tests and IgE-RAST--as a further criterion for deciding to stop venom therapy if it turns negative with the washed cells.

[The basophil degranulation test in the diagnosis of Hymenoptera sting allergy]. / Der Basophilen-Degranulationstest in der Diagnostik der Hymenopterengift-Allergie.

In 98 patients with a suspected immediate type allergy to hymenoptera stings, skin tests (ST) with venom extracts, RAST determinations of venom specific serum IgE antibodies and basophil degranulation tests (BDT) with unwashed leukocytes were performed. BDT is a technically simple in-vitro method for detecting cell fixed IgE without risk for the patient. The specificity of BDT was investigated in a group of 10 controls not allergic to insect stings, of whom 7 were atopics. A good correlation could be demonstrated between BDT and the histamine release test (HRT), which was performed in the Institute for Allergy and Clinical Immunology, Inselspital, Bern. For the detection of a sensitization to insect venoms, ST with venom extracts were the most sensitive, followed by RAST and BDT; in patients whose allergic sting reactions dated back some time BDT was more frequently positive than RAST, which measures excessive serum IgE and not cell bound IgE responsible for the clinical symptomatology. As BDT--in the presence of patient serum--reflects the influences of different humoral (specific IgE, specific IgG) and cellular factors ("releasability"), it seems more appropriate to the actual clinical allergy situation than ST or RAST. Some case reports are presented to illustrate this. However, BDT does not represent an alternative to ST or RAST but a significant complement in diagnostically unclear cases and an aid in deciding to start specific venom immunotherapy, if history, ST and RAST are not sufficient. An appropriate score system ist proposed.

[Value of the basophil degranulation test (BDT) in the follow-up of immunotherapy with hymenoptera venoms]. / Stellenwert des Basophilen-Degranulationstestes (BDT) als Verlaufskontrolle einer Immuntherapie mit Hymenopterengiften.

The basophil degranulation test (BDT) with the plasma of 24 patients undergoing venom immunotherapy was usually negative after only 6 months of treatment. Cellular sensitivity, however, could still be proved by BDT on purified cells, i.e. after removal of the plasma which contained blocking IgG antibodies. In 30% of the cases, we found primary desensitization of the basophils, which means that the IgG antibodies did not have any influence on the test results.

Treatment of cystinosis with cysteamine. A pilot study determining dose and form of application.

A pilot study with cysteamine treatment was performed in three children with the nephropathic form of cystinosis. Two children underwent renal transplantation shortly before treatment. The aim of the study was to find a practicable form of application and a corresponding effective dose. Cysteamine in gelatine capsules together with 0.2% silicic acid as a dessicator turned out to be the most acceptable galenic form, compared to sirup or suppositories. Among three dosage regimens, the dosage of 50 mg/kg/day is effective as judged by the leucocyte cystine content, even if given in only three doses per day. No side effects of the cysteamine treatment (even at a dose of 90 mg/kg/day) were noted. Whether this treatment is preventing progression of disease will have to be examined either in transplanted patients by measuring non-renal parameters or in very young infants with cystinosis whose kidneys are not damaged yet.

The role of enzyme variants, polymorphisms and enzyme hybrids in enzyme deficiency conditions.

Based on the heterogeneity observed in two red cell enzymes, i.e. glucose 6-phosphate dehydrogenase (E.C. 1.1.1.49) and catalase (E.C.1.11.1.6), the role of different types of enzyme variants in enzyme deficiency conditions is discussed. For theoretical and practical reasons variants of unusually low specific activity and of low stability have to be distinguished. Whereas in the former type the activity level in blood more or less reflects the situation in other tissues, this is not the case for unstable mutants, e.g. the enzyme variant found in Swiss-type acatalasemia. In heterozygous carriers the situation can be complicated by the fact that variants of oligomer enzymes (e.g. catalase) are present as molecular hybrids exerting almost normal stability and activity.

The microheterogeneity of human plasma alpha1-acid glycoprotein.

alpha1-Acid glycoprotein was isolated in the homogeneous state from the plasma of 33 normal individuals and subjected to analytical isoelectric focusing before and after treatment with neuraminidase. The native glycoprotein preparations, resolved into 6 to 8 bands, were quantitated and grouped into two classes according to the patterns obtained: One class exhibited a relatively anodic and the other a relatively cathodic distribution of the protein bands. The isoelectric points of these bands ranged from pH 2.90 to 3.30. After treatment with neuraminidase the resulting asialo-glycoproteins were also quantitated and afforded two types of fundamentally different patterns from those mentioned above, namely one type with one and the other type with two main bands and both exhibiting several minor components. The isoelectric points of the main bands were found to be of pH 4.55 and 4.70 while those of the minor bands were at both the anodic and cathodic side of the major bands. No apparent relationship between the patterns of the native and those of the asialo-glycoproteins could be established. In addition, a new variant was noted whose major band focused at a pH of 5.0. The microheterogeneity of alpha1-acid glycoprotein is thus interpreted to be due to the amino acid replacements of this protein in combination with the linkages of the sialyl to the galactosyl residues in the native protein.

Protection of human neutrophils by endogenous catalase: studies with cells from catalase-deficient individuals.

To investigate the importance of catalase as a protecting enzyme against oxidative damage in phagocytic leukocytes, we have tested the functional capacity of neutrophils from two individuals homozygous for Swiss-type acatalasemia and from two individuals heterozygous for this deficiency. In the former cells, 25-30% of residual activity of catalase was present. In the latter cells, the values were close to normal. Chemotaxis towards casein, release of lysosomal enzymes and hydrogen peroxide during phagocytosis of zymosan, and intracellular killing of Staphylococcus aureus were normal in all cells tested. Inhibition of heme enzymes with azide (2 mM) enhanced the respiration and hexose monophosphate shunt activity of normal, but not of homozygous acatalasemic, neutrophils. This indicates that the enhancement in normal cells is, at least in part, due to catalase inhibition. After 15 min preincubation with an H(2)O(2)-generating system (glucose plus glucose oxidase), the respiratory response to zymosan phagocytosis was strongly depressed in the homozygous acatalasemic and in normal, azide-treated neutrophils, but not in normal, untreated cells. Under these conditions, the release of lysosomal enzymes was depressed and that of lactate dehydrogenase enhanced, in catalase-deficient and in catalase-inhibited, but not in normal, neutrophils. During prolonged incubation with the H(2)O(2)-generating system (30-60 min), the reduction level of intracellular glutathione remained high and the hexose monophosphate shunt continued to operate normally in all cells tested. Thus, although the function of neutrophils without catalase activity was depressed by extracellular hydrogen peroxide, the H(2)O(2) degradation via the glutathione redox system remained operative. The results indicate that the glutathione redox system by itself efficiently protects phagocytosing neutrophils against their own oxidative products. During heavy external oxidative stress, however, both catalase and the glutathione redox system are needed for adequate protection.

Catalase and dehydroascorbate reductase in human polymorphonuclear leukocytes (PMN): possible functional relationship.

In leukocytes (PMN) of individuals with Swiss type acatalasemia, the rate of dehydroascorbate reduction is 4 times normal. This observation suggests that the protective function served by catalase in human PMN is supported by dehydroascorbate reductase.