Inhibition of TGF-beta modulates macrophages and vessel maturation in parallel to a lowering of interstitial fluid pressure in experimental carcinoma

A pathologically elevated interstitial fluid pressure (IFP) is a characteristic of both clinical and experimental carcinoma. The soluble TGF-beta receptor type II-murine Fc:IgG2A chimeric protein (Fc:TbetaRII) lowers IFP in the KAT-4 experimental model for anaplastic thyroid carcinoma. Analyses of messenger RNA (mRNA) expressions by Affymetrix microarrays and RNase protection assays, as well as of protein expressions identified tumor macrophages as targets for Fc:TbetaRII. Treatment with Fc:TbetaRII reduced albumin extravasation, increased coverage of alpha-smooth muscle actin-positive cells and reduced expression of NG2, a marker of activated pericytes, in KAT-4 carcinoma blood vessels. Specific inhibition of interleukin-1 (IL-1), a major cytokine produced by activated macrophages, lowered carcinoma IFP to a similar degree as Fc:TbetaRII but had no significant effect on the parameters of blood vessel maturation. Neither Fc:TbetaRII nor inhibition of IL-1 changed blood vessel density. Finally, pretreatment of KAT-4 carcinomas with Fc:TbetaRII increased the antitumor efficacy of doxorubicin. Our data emphasize a potential role of tumor macrophages in carcinoma physiology and identify these cells as potential stromal targets for treatment aimed to improve efficacy of chemotherapy.     

Effect of the external donors on the polymerization of 4-methyl-1-pentene with high activity MgCl2/TiCl4 catalytic system

In order to find out correlations between the structure of an external donor and the obtained polymer, the effects of different external donors on the activity and stereospecificity of the MgCl₂/TiCl₄ catalytic system in the bulk polymerization of 4-methyl-1-pentene (4MP) were carefully studied. Different silane compounds of the structure R_nSi(OR')_4-n (where: n = 1–3, R = alkyl/phenyl, R = alkyl) and Al(i-Bu)₃ (TIBA) were used as external donors and cocatalyst, respectively. The effect of the donor/TIBA mole ratio on the activity and stereospecificity of the catalytic system was studied. Some major effects were observed for the three different external donors, namely, Me₃Si(OMe), Me₂Si(OMe)₂, and Me₃Si(OBu)₃, in the 4MP polymerization process. It was observed that the effect of the external silane donor on the polymerization strongly depends upon the size of the alkoxy and hydrocarbon (alkyl/phenyl) groups which are attached to the silicon atom. A selective deactivation of the non-stereospecific centers, as well as a transformation of the non-stereospecific into isospecific centers, is assumed to occur. On the basis of the obtained results and literature data available for the propene polymerization, the concept of structural conformity between the ligand-surrounded active center and the monomer molecule was carried forward.     

Strukturelle Effekte in Systemen MnLi/Butadien/Styrol

The structure of the butadiene unit C₄H₆ in the hydrolysis products of the oligomers RM_nC₄H₆M_m'Li and RM_nC₄H₆Li (where R is sec-butyl, M and M' are perdeuterobutadiene or styrene) was estimated by the use of IR- and NMR-spectroscopy. The dependency of the C₄H₆-structure on the nature of both the preceding unit M and the attacking monomer M' was investigated. The variation of the concentration of the organolithium compounds during the oligomer synthesis on the one hand, and during their hydrolysis on the other hand, showed a dependency of the C₄H₆-structure on the conditions of the respective experiments.     

In vitro stimulation of antibody formation by peritoneal cells: III. Effect of active immunization on the subsequent in vitro performance of peritoneal and spleen cells

Male CBA mice were given a single intraperitoneal injection of sheep red blood cells (SRBC) or horse red blood cells (HRBC). They were killed at intervals of 1–10 days thereafter, and micro-cultures of spleen cells or peritoneal cells (PC) were prepared. These consisted of a thin film of tissue culture medium containing carboxymethyl cellulose (CMC), mouse lymphoid cells, guinea-pig complement and either SRBC or HRBC, held at 37° under liquid paraffin. Cultures were read repeatedly for appearance of haemolytic plaques.

PC from SRBC-immunized mice showed an altered reactivity on SRBC monolayer cultures. The peak plaque count achieved in vitro fell progressively for 4 days after immunization, and then returned to normal by day 7. The actinomycin D resistant component of the PC response rose rapidly; at 1 day after immunization it was equal to the total response. Over the next 3 days after immunization it fell again to normal levels. The results suggested that the in vivo injection sets in train events locally in the peritoneal cavity which resembled those following in vitro culture of normal PC in SRBC monolayers. The effects were immunologically specific as only marginal changes followed the injection of HRBC.

Spleen cells from SRBC-immunized mice, when cultured in SRBC monolayers, yielded many cells capable of giving plaques after 5–60 minutes incubation, as expected. These were deemed to be cells forming antibody at the moment of killing of the animal. In addition, such cultures developed new plaques over the subsequent 23 hours in culture. These were produced by cells not initially forming antibody which switched into antibody secretion at some time during culture. At early time points after immunization, this second type of cell was much more numerous than the first type. The switch from non-secretor status could occur in the presence of a high concentration of actinomycin D. Operationally these non-secretors in immunized spleens resembled an important fraction of PC from unimmunized retired breeder mice. The progressive conversion of non-secretor cells into secretors, if it occurs in vivo, would have a major influence on the kinetics of appearance of PFC in a spleen after immunization.

While spleen cells from mice immunized with HRBC performed on HRBC monolayers much as described above, PC from HRBC-immunized mice could not be induced to cause significant lysis in HRBC monolayers. The same was true of PC from mice chronically fed with HRBC. In fact, no method has yet been found to persuade PC to produce lytic plaques active against erythrocytes other than SRBC.

     

Intertubular growth in pure seminomas: associations with poor prognostic parameters

Clinical stage I seminomas are effectively treated with surgery raising concerns as to when to give adjuvant radiation therapy given the risk of secondary malignancies. A recent randomized trial found tumor size and rete testis invasion to be the strongest predictors of relapse in clinical stage I seminomas. These 2 parameters may be surrogate measures of tumor volume. Intertubular seminoma (ITS) of the testis describes the presence of neoplastic germ cells within the interstitium of the testis. These cells are detected away from the main macroscopic mass. Because ITS can infiltrate in a 3-dimensional fashion, it may also represent a measure of tumor volume not usually noted in standard pathology reporting. The goal of this study was to determine the incidence of ITS in pure seminomas and its association with other prognostic parameters. One hundred twenty consecutive pure seminomas surgically removed between 1998 and 2003 were evaluated. ITS was defined as the presence of an interstitial or intertubular growth pattern of tumor cells, which was noncontiguous with the main tumor and present at least 3 high-power fields away from the tumor mass. The average tumor size was 3.4 cm. Of the entire cohort of patients, which included pathological stages T1 through T3, 11% had invasion through the tunica albuginea, 51% had rete testis invasion, 51% had lymphovascular invasion, 93% had associated intratubular germ-cell neoplasia, and 36% had ITS. ITS was significantly associated with rete testis invasion ( P = .001). Logistic regression analysis looking at ITS, tumor size, patient age, and lymphovascular invasion revealed that only ITS was associated with rete testis invasion (RR, 4.1, P < .0001). ITS is present in a significant proportion of pure seminomas and has a significant association with rete testis invasion. The presence of ITS may therefore be an important prognostic factor, not only because it alters the calculated size of the tumor but also because it has an association with rete testis invasion.     

Regulator T Cells: Specific for Antigen and/or Antigen Receptors?

Adaptive immune responses are regulated by many different molecular and cellular effectors. Regulator T cells are coming to their rights again, and these T cells seem to have ordinary α/β T‐cell receptors (TCRs) and to develop in the thymus. Autoimmune responses are tightly regulated by such regulatory T cells, a phenomenon which is beneficial to the host in autoimmune situations. However, the regulation of autoimmune responses to tumour cells is harmful to the host, as this regulation delays the defence against the outgrowth of neoblastic cells. In the present review, we discuss whether regulatory T cells are specific for antigen and/or for antigen receptors. Our interest in these phenomena comes from the findings that T cells produce many more TCR‐α and TCR‐β chains than are necessary for surface membrane expression of TCR‐αβ heterodimers with CD3 complexes. Excess TCR chains are degraded by the proteasomes, and TCR peptides thus become available to the assembly pathway of major histocompatibility complex class I molecules. Consequently, do T cells express two different identification markers on the cell membrane, the TCR‐αβ clonotype for recognition by B‐cell receptors and clonotypic TCR‐αβ peptides for recognition by T cells?     

Analysis of DNA ligase IV mutations found in LIG4 syndrome patients: the impact of two linked polymorphisms

LIG4 syndrome patients have hypomorphic mutations in DNA ligase IV. Although four of the five identified patients display immunodeficiency and developmental delay, one patient was developmentally normal. The developmentally normal patient had the same homozygous mutation (R278H) in DNA ligase IV as one of the more severely affected patients, who additionally had two linked polymorphisms. Here, we examine the impact of the mutations and polymorphisms identified in the LIG4 syndrome patients. Examination of recombinant mutant proteins shows that the severity of the clinical features correlates with the level of residual ligase activity. The polymorphisms decrease the activity of DNA ligase IV by approximately 2-fold. When combined with the otherwise mild R278H mutation, the activity is reduced to a level similar to other LIG4 patients who display immunodeficiency and developmental delay. This demonstrates how coupling of a mutation and polymorphism can have a marked impact on protein function and provides an example where a polymorphism may have influenced clinical outcome. Analysis of additional mutational changes in LIG4 syndrome (R580X, R814X and G469E) have led to the identification of a nuclear localization signal in DNA ligase IV and sites impacting upon DNA ligase IV adenylation.     

Induction of biologically active antineutrophil cytoplasmic antibodies by immunization with human apoptotic polymorphonuclear leukocytes

Translocation of intracellular components to the cell surface during the priming or apoptosis of polymorphonuclear leukocytes (PMN) is an important mechanism for interaction of antineutrophil cytoplasmic antibodies (ANCA) with these antigens. To test the capacity of apoptotic PMN to trigger production of ANCA, six groups of mice were immunized with either live or apoptotic lymphocytes, or with live, apoptotic, formalin-fixed, or lysed PMN. Mice immunized with both live and apoptotic neutrophils developed high titers of antibodies which gave a granular cytoplasmic immunofluorescent pattern. These antibodies were specific for lactoferrin and myeloperoxidase. Following a second intravenous infusion of apoptotic PMNs, mice developed anti-PR3 antibodies. Vasculitis lesions were not found in mice which developed ANCA. The ANCA-containing IgG fraction induced superoxide production by human PMNs. These results support the hypothesis that neutrophil-specific antigens presented on the cell membranes of apoptotic PMN may induce ANCA in the proper conditions.     

Evidence for a change in the expression of beta2-microglobulin-assoicated membrane structures on leukaemic human cells.

Cell-associated and serum beta2-microglobulin was estimated in seven patients with chronic lymphocytic leukaemia. The amount of cell-associated beta2-microglobulin was significantly reduced (P less than 0.01), due to a decrease in the fraction of beta2-microglobulin that passes unretarded through a concanavalin A affinity column (presumably non-HLA-associated beta2-microglobulin). Serum concentrations of beta2-microglobulin were increased, but no correlation was found between the decrease in cell-associated beta2-microglobulin and the increase in serum beta2-microglobulin. All of the beta2-microglobulin from leukaemic serum was eluted corresponding to a molecular weight of 11,800 and none of it was retarded on a concanavalin A affinity column. The decrease in cell-associated beta2-microglobulin might reflect a change in the qualitative or quantitative expression of beta2-microglobulin-associated membrane structures on the leukaemic cells, perhaps conferring resistance to the cells against hypothetical immunological host defence mechanisms.