Mast cells decrease efficacy of anti-angiogenic therapy by secreting matrix-degrading granzyme B.

Resistance towards VEGF-centered anti-angiogenic therapy still represents a substantial clinical challenge. We report here that mast cells alter the proliferative and organizational state of endothelial cells which reduces the efficacy of anti-angiogenic therapy. Consequently, absence of mast cells sensitizes tumor vessels for anti-angiogenic therapy in different tumor models. Mechanistically, anti-angiogenic therapy only initially reduces tumor vessel proliferation, however, this treatment effect was abrogated over time as a result of mast cell-mediated restimulation of angiogenesis. We show that mast cells secrete increased amounts of granzyme b upon therapy, which mobilizes pro-angiogenic laminin- and vitronectin-bound FGF-1 and GM-CSF from the tumor matrix. In addition, mast cells also diminish efficacy of anti-angiogenic therapy by secretion of FGF-2. These pro-angiogenic factors act beside the targeted VEGFA-VEGFR2-axis and reinduce endothelial cell proliferation and angiogenesis despite the presence of anti-angiogenic therapy. Importantly, inhibition of mast cell degranulation with cromolyn is able to improve efficacy of anti-angiogenic therapy. Thus, concomitant mast cell-targeting might lead to improved efficacy of anti-angiogenic therapy.Resistance towards VEGF-centered anti-angiogenic therapy is an important clinical challenge. Here, the authors show that mast cells mediate resistance to anti-angiogenetic inhibitors by altering the proliferative and organizational state of endothelial cells through mobilization of FGF-1 and GM-CSF from the tumor matrix and secretion of FGF-2.

Acute inhibition of central c-Jun N-terminal kinase restores hypothalamic insulin signalling and alleviates glucose intolerance in diabetic mice.

The hypothalamus has been identified as a main insulin target tissue for regulating normal body weight and glucose metabolism. Recent observations suggest that c-Jun-N-terminal kinase (JNK)-signalling plays a crucial role in the development of obesity and insulin resistance because neuronal JNK-1 ablation in the mouse prevented high-fat diet-induced obesity (DIO) and increased energy expenditure, as well as insulin sensitivity. In the present study, we investigated whether central JNK inhibition is associated with sensitisation of hypothalamic insulin signalling in mice fed a high-fat diet for 3 weeks and in leptin-deficient mice. We determined whether i.c.v. injection of a pharmacological JNK-inhibitor (SP600125) improved impaired glucose homeostasis. By immunohistochemistry, we first observed that JNK activity was increased in the arcuate nucleus (ARC) and the ventromedial hypothalamus (VMH) in both mouse models, relative to normoglycaemic controls. This suggests that up-regulation of JNK in these regions is associated with glucose intolerance and obesity, independent of leptin levels. Acute i.c.v. injection of SP600125 ameliorated glucose tolerance within 30 min in both leptin-deficient and DIO mice. Given the acute nature of i.c.v. injections, these effects cannot be attributed to changes in food intake or energy balance. In a hypothalamic cell line, and in the ARC and VMH of leptin-deficient mice, JNK inhibition by SP600125 consistently improved impaired insulin signalling. This was determined by a reduction of phospho-insulin receptor substrate-1 [IRS-1(Ser612)] protein in a hypothalamic cell line and a decline in the number of pIRS-1(Ser612) immunoreactive cells in the ARC and VMH. Serine 612 phosphorylation of IRS-1 is assumed to negatively regulate insulin signalling. In leptin-deficient mice, in both nuclei, central inhibition of JNK increased the number of cells immunoreactive for phospho-Akt (Ser473) and phospho-GSK-3ß (Ser9), which are important markers of insulin signalling. Collectively, our data suggest that the acute inhibition of central JNK improves impaired glucose homeostasis and is associated with sensitisation of hypothalamic insulin signalling.

Increase in immunoglobulin M antibodies against gut bacteria during acute hepatitis A.

The marked increase in the total serum immunoglobulin M (IgM) is a characteristic feature of acute hepatitis A. To study the nature of this IgM, we assayed serial titers of IgM antibodies against various antigens during and after acute hepatitis A. The antibodies against blood group antigen remained unchanged throughout the observation period. Thus, the production or metabolism of IgM was not nonspecifically altered. The IgM antibody against hepatitis A antigen decreased and finally disappeared during convalescence as expected. However, its time course did not correlate quantitatively with the concentration of the total serum IgM. In contrast, IgM antibodies against gut bacteria Bacteroides fragilis and Streptococcus faecalis were considerably elevated in all patients at the onset of the disease, and they normalized similarly to the total IgM during convalescence. IgM antibodies against Escherichia coli were elevated only in some of the patients. The data suggest that the amount of IgM antibodies against gut bacteria contributes significantly to the increase in the total serum IgM in acute hepatitis A.

Kinetics, subtype specificity and immunoglobulin class of anti-HBs induced by hepatitis B vaccine.

The protective effect of anti-HBs against hepatitis B virus is proven only for the common antibody anti-HBs/a but not for subtype specific antibody. Using subtype specific radioimmunoassays, anti-HBs/a and anti-HBs/d were quantitated in recipients of an HBsAg/ad vaccine. All persons developed anti-HBs/a. The relative proportion of anti-HBs/d was variable and very high at the beginning of the immune response. At this time the anti-HBs was predominantly in the IgM class. IgM-anti-HBs disappeared rapidly after its peak value and was more slowly replaced by IgG-anti-HBs. Persons who had only anti-HBs or anti-HBc as the only antibody did usually not react with an anamnestic booster response and developed IgM- anti-HBs after vaccination. An injection schedule of 0, 1, 4 months produced ten times higher titers than a 0, 1.5, 3 months schedule 4 weeks after the third injection. However, 6 months later titers were essentially identical. Nine of ten "non-responders" became positive after a fourth injection.

[Differing results of direct and indirect solid phase radioimmunoassay for HBsAg in acute hepatitis (author's transl)]. / Unterschiedliches Ergebnis des direkten und indirekten Festphasenradioimmuntests zum Nachweis von HBsAg bei akuter Hepatitis.

In 54 patients suffering from acute viral hepatitis the indirect solid phase radioimmunoassay (ind-SPRIA) for HBsAg was positive in 9 cases the direct solid phase radioimmunoassay (d-SPRIA) being negative. In 2 further cases ind-SPRIA was positive during several weeks but d-SPRIA only once. AntiHBc could be detected in 9 of these patients. In 7 patients the usual decrease of the transaminase activity was followed by a second elevation with prolongation of disease. The unknown factor detected by ind-SPRIA suggests a special form of acute hepatitis.

Experimental non-A, non-B hepatitis: four types of cytoplasmic alteration in hepatocytes of infected chimpanzees.

On the occasion of an outbreak of non-A, non-B hepatitis in a plasmapheresis centre (81 cases, incubation period: 3--6 weeks) a pool of 12 plasma samples was obtained in the early phase of increasing transaminases. Two chimpanzees were inoculated, each receiving 12 ml of the pooled plasma. After an incubation period of 10--12 weeks a mild non-A, non-B hepatitis developed. Serum transaminases were slightly elevated. Needle biopsies, taken fortnightly, showed a slight activation of Kupffer cells (6--8 weeks), single cell necroses, and infiltration of the portal tracts (10--13 weeks). Electron microscopically four types of cytoplasmic change, were found in hepatocytes and assumed to be specific for the infection, Type I: Sponge-like inclusion (6 weeks after inoculation) composed of a dense matrix and irregularly arranged membranes. Type II: Attaching curved membranes (8 weeks), developing by close apposition of two cisternae of smooth endoplasmic reticulum. Type III: Cylindrical complexes (10 weeks), already described in literature. Type IV: Microtubular aggregates, usually neighbouring type III structures. The findings suggest 1) that the agent of the present infection is, at least in part, identical with that of the long incubation type of experimental non-A, non-B hepatitis, and 2) that ultrastructural alterations may precede manifest hepatitis.

[Preparation and testing of a hepatitis B vaccine (author's transl)]. / Herstellung und Erprobung eines Hepatitis-B-Impfstoffes.

Starting with 41.5 l of plasma from anti-HBe positive carriers of HBs antigen, 11,400 doses of a hepatitis B vaccine with 42 micrograms HBsAg-protein and 11,300 national units HBsAg activity per dose were obtained. After purification, HBsAg is obtained in 99% purity with a yield of more than 90% protein. A possible residual infectivity was inactivated by a diluted formalin solution. The infectivity test in chimpanzees confirmed the absence of infectious hepatitis viruses (HBV and nonA-nonB). In guinea pigs the immunogenicity of the vaccine was comparable to that of the reference preparation from the U.S. National Institute of Health. The presence of Al(OH)3 in the vaccine increased the anti-HBs titre by factors of 30-50. After vaccination with two doses 41 of 45 persons became anti-HBs positive, with three doses 42 of 45 persons developed anti-HBs. Median anti-HBs titre after the third doses: schedule I (three doses in intervals of 6 weeks) 427 mWHO-U/ml; schedule II (two doses at an interval of 4 weeks, third doses 4 months after the first doses) 1535 mWHO-U/ml. The vaccine was well tolerated. There were minor local reactions only.

Safety and potency aspects in the preparation of an experimental HBsAg vaccine.

No experimental setting is available to exclude residual infectivity in HBsAg vaccines derived from human plasma. Thus, safety can be achieved only by means of their preparation. To reduce infectivity of the starting material, only plasma from healthy anti-HBe positive donors was used. In the FRG, 50% of all healthy HBsAg carriers with anti-HBe have a suitable serum level of 5 to 20 micrograms/ml. The purification procedure removed hepatitis B virus by a factor greater than 10(4). The purified product contained only the HBsAg proteins and no serum protein, as shown by SDS gel electrophoresis. The pure HBsAg was treated with formalin 1:500 at 37 degrees C for 4 days. A loss of 30 to 50% antigenicity was tolerated to achieve the highest possible destruction of known and unknown infectious agents. After inactivation, the HBsAg was bound to aluminium hydroxide gel. The gel was washed repeatedly to remove the formalin. Doses of 40 micrograms or 20 micrograms absorbed HBsAg protein were given to greater than 2500 persons without serious side effects. In greater than 97% anti-HBs was formed with a median titer of 1900 I.U./ml.