Expression and intracellular localization of the human N-acetylmuramyl-L-alanine amidase, a bacterial cell wall-degrading enzyme

N-acetylmuramyl-L-alanine amidase (NAMLAA) specifically degrades peptidoglycan, which is a major component of bacterial cell walls with strong inflammatory properties. For instance, peptidoglycan is capable of stimulating peripheral blood cells to release pro-inflammatory cytokines and is capable of inducing chronic arthritis in an animal model. In a previous study we found that degradation of peptidoglycan by purified NAMLAA reduced its inflammatory effects. To determine where NAMLAA is located in tissues, monoclonal antibodies against purified NAMLAA were produced for use in immunohistochemistry, immunoelectron microscopy, flow cytometric analysis, and Western blotting. The immunohistochemical studies showed NAMLAA-positive cells in human spleen, liver, arthritic synovial tissues, and lymph nodes. In flow cytometric studies of blood and bone marrow, neutrophilic and eosinophilic granulocytes proved to be positive. Monocytes were negative, although they do contain lysozyme, the other important peptidoglycan-degrading enzyme. However, mature macrophages obtained by bronchoalveolar lavage and subsequent selection based on autofluorescence did possess NAMLAA. In immunocytochemical staining of blood smears, thrombocytes were also positive for NAMLAA. Western blot analysis and immunoelectron microscopy of neutrophils and eosinophils showed that NAMLAA is located in azurophilic granules of neutrophils and in secretory vesicles and crystalloid-containing granules of eosinophils. Flow cytometric analysis of blood and bone marrow from different French-American-British-classified acute myeloid leukemia (AML) patients showed that AML-M2 myeloblasts were the first in the granulocyte maturation lineage that were positive for NAMLAA. The more immature AML, such as AML-M0 and AML-M1, did not express NAMLAA. CD15- and CD13-negative megakaryoblasts, corresponding to AML-M7, were also positive for NAMLAA. The expression pattern of NAMLAA in the myeloid lineage suggests that the monoclonal antibody AAA4, recognizing NAMLAA, is useful for discrimination between AML in the monocyte lineage and in the granulocyte lineage.     

Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors

This study shows that induction of tumor-specific CD4+ T cells by vaccination with a specific viral T helper epitope, contained within a synthetic peptide, results in protective immunity against major histocompatibility complex (MHC) class II negative, virus-induced tumor cells. Protection was also induced against sarcoma induction by acutely transforming retrovirus. In contrast, no protective immunity was induced by vaccination with an unrelated T helper epitope. By cytokine pattern analysis, the induced CD4+ T cells were of the T helper cell 1 type. The peptide-specific CD4+ T cells did not directly recognize the tumor cells, indicating involvement of cross-priming by tumor-associated antigen-presenting cells. The main effector cells responsible for tumor eradication were identified as CD8+ cytotoxic T cells that were found to recognize a recently described immunodominant viral gag-encoded cytotoxic T lymphocyte (CTL) epitope, which is unrelated to the viral env-encoded T helper peptide sequence. Simultaneous vaccination with the tumor-specific T helper and CTL epitopes resulted in strong synergistic protection. These results indicate the crucial role of T helper cells for optimal induction of protective immunity against MHC class II negative tumor cells. Protection is dependent on tumor-specific CTLs in this model system and requires cross-priming of tumor antigens by specialized antigen-presenting cells. Thus, tumor-specific T helper epitopes have to be included in the design of epitope-based vaccines.     

Activation or frustration of anti-tumor responses by T-cell-based immune modulation

Cataleptogenic effects of haloperidol (1 mg/kg i.p.) in rats was antagonized by caffeine and theophylline (10-50 mg/kg i.p.), and by selective adenosine A2 receptor antagonist (3,7-dimethyl-1-propargylxanthine) (3 and 6 mg/kg i.p.). Selective A1-adenosine receptor antagonist (8-cyclopentyltheophylline) (1.5 and 3 mg/kg i.p.) was not able to reduce this effect of haloperidol. These results confirm the antagonistic interaction between adenosine A2A and dopamine D2 receptors, and suggest the involvement of adenosine A2 receptors in the mechanisms of catalepsy.     

Peptide vaccination with an anchor-replaced CTL epitope protects against human papillomavirus type 16-induced tumors expressing the wild-type epitope

PURPOSE: Upper-extremity thrombosis appears to be more frequent today, comprising about 2% of all deep venous limb thrombosis. Its severity depends on the type of possible complications, i.e., pulmonary embolism and post-thrombotic sequelae. In this retrospective series, we investigated both the predisposing factors and the evolution of upper-extremity deep venous thrombosis. METHODS: Forty-nine consecutive patients (24 men and 25 women, mean age 50.2 years) with upper extremity deep venous thrombosis documented by color Doppler ultrasonography (n = 47) or phlebography (n = 2) were included in the study. RESULTS: Clinical manifestations were mainly pain (81.6%) and edema (93.9%). Mean time between the onset of clinical signs and diagnosis was 7.2 days. Thrombosis involved humeral (26.5%), axillary (46.9%), subclavian (73.5%) and jugular (24.5%) veins. Causative factors were malignancies (32.7%), venous catheters (22.4%), deep venous thrombosis related to effort or thoracic outlet syndrome (22.5%) and thrombophilic states (8.2%). During the 6-month follow-up, six patients developed symptomatic pulmonary embolism (12.2%); one recurrence (2.2%) and 19 post-thrombotic sequelae such as residual edema (36.7%) were also observed. Initial therapy included heparin administration, principally subcutaneous low molecular weight heparins (n = 36/49). CONCLUSION: This series highlights the fact that upper-extremity deep venous thrombosis is mainly secondary to either malignancies or catheterization. Moreover, it confirms that color Doppler ultrasonography may be useful in the diagnosis of the disease and also underlines the high frequency of severe complications, i.e., pulmonary embolism and post-thrombotic sequelae. Finally, this study also demonstrates that low molecular weight heparins should be considered as the initial treatment of choice.     

Analysis of sea spikes in radar sea clutter data

In this paper three sets of high-resolution, coherent, and polarimetric radar sea clutter data are analyzed and compared with radar sea clutter models. The nature of the data allows a thorough analysis of the power, polarization and velocity of the sea clutter. It is shown that these quantities, especially the velocity, are good measures of many physical properties of the ocean surface. Furthermore, it is shown that these physical properties match well with the sea clutter models. Sea clutter is found to consist of two components, a diffuse background, characterized by low values of backscattered power, HH/VV polarization ratio and Doppler velocity, and a number of spiking events, which possess higher power, polarization ratio and velocity. The background is reasonably well modeled by tilt-modulated Bragg scattering, whereas the spikes may be associated with the scattering on steepened and/or breaking waves. Moreover, it is shown that the influence of microbreakers has to be taken into account to explain the relatively high polarization ratio. A breaking wave origin for the spikes is supported in two ways. First, by a detailed analysis of the temporal behavior of individual spike backscatter properties, and second, by a statistical analysis of the entire population of spikes.     

Immunization with human papillomavirus type 16 (HPV16) oncoprotein-loaded dendritic cells as well as protein in adjuvant induces MHC class I-restricted protection to HPV16-induced tumor cells

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical cancer. In this study, we demonstrate that dendritic cells (DCs) pulsed with HPV16 E7 protein are not only recognized in vitro by E7-specific CTLs but also elicit E7-specific CTL responses in vivo, associated with protection against a challenge with syngeneic HPV16-induced tumor cells. Vaccination with soluble E7 protein in incomplete Freund's adjuvant likewise induces E7-specific CTL responses associated with tumor protection. The presence of HPV16 E7-specific CTLs in vivo and the observation that depletion of CD8+ cells completely abolishes tumor protection demonstrate that CTLs are the major effector cells in mediating antitumor activity. The in vivo involvement of DCs in the activation of protective CTLs is suggested by the surface display of E7 peptide-loaded MHC class I molecules on these cells after E7 protein immunization. These data show that HPV16 E7 protein-pulsed DCs, as well as the administration of E7 protein antigen in adjuvant, can effectively stimulate tumor-specific MHC class I-restricted CD8+ T-cell-mediated protective immunity to HPV16-induced cancers.     

Efficient tumor eradication by adoptively transferred cytotoxic T-cell clones in allogeneic hosts

Tumor-specific cytotoxic T cells (CTLs) can play an important role against cancer as illustrated by the observation that adoptive transfer of tumor-specific CTLs can mediate potent anti-tumor effects. Although such CTLs can be detected at the tumor site, relatively little is known about the mechanisms by which they enter the tumor. In this study, the role of major histocompatibility complex (MHC) class 1 molecules on vascular endothelium in the tumor in entry of, and tumor eradication by, tumor-specific CTL was investigated. Two H-2Db-restricted CTL clones recognizing peptide VNIRNCCYI on human adenovirus type 5 early region 1-(Ad5E1)-induced tumors were used to test whether CTLs were able to cross the vascular endothelium lacking the restricting MHC molecule. One CTL clone recognizes peptide VNIRNCCYI in the context of both H-2Db and H-2Dbm14 molecules. The other CTL clone recognizes this peptide only in the context of H-2Db. Adoptive transfer of these CTLs leads to eradication of Ad5E 1-induced, H-2Db-expressing tumors in B6(H-2Db+) and Bm14(H-2Db-) nude mice. Our data show that presentation of tumor-derived peptides by MHC molecules on endothelial cells of blood vessels in a tumor do not play a major role in eradication of tumors by adoptively transferred CTL in combination with interleukin-2. Moreover, our data show that successful adoptive CTL immunotherapy is possible across allogeneic barriers, without inducing severe side effects, provided the tumor expresses the MHC class 1-peptide complex recognized by the CTLs.     

Cross-priming of CTL responses in vivo does not require antigenic peptides in the endoplasmic reticulum of immunizing cells

It has been proposed that the cross-priming of CTL responses in vivo involves the transfer to host APCs of heat shock protein glycoprotein 96-chaperoned antigenic peptides released from the endoplasmic reticulum (ER) of dying or infected cells. We have tested this possibility directly using TAP-deficient cell lines lacking antigenic ER peptides derived from two model Ags, the human adenovirus type 5 early regions E1A and E1B. Although both proteins were well expressed, the cells were not recognized by E1A- or E1B-specific CTLs unless the relevant epitope was either provided exogenously as a synthetic peptide or targeted to the ER in a TAP-independent fashion. Despite the absence of these ER peptides, the TAP1-/- cells were able to efficiently cross-prime E1A- and E1B-specific CTLs following immunization of syngeneic mice. These results indicate that, although purified peptide/glycoprotein 96 complexes are potent immunogens, the mechanism of CTL cross-priming in vivo does not depend upon antigenic peptides in the ER of immunizing cells.     

Efficient direct priming of tumor-specific cytotoxic T lymphocyte in vivo by an engineered APC

Although numerous studies have documented a role for B7-1 (CD80) in the induction of antitumor CTL immunity, it is presently unclear to what extent expression of this costimulatory molecule truly endows tumors with significant in vivo APC (antigen-presenting cell) capacity. Recent studies have, in fact, demonstrated that cross-priming, rather than direct priming, may constitute the major mechanism of CTL induction by B7-1 expressing tumors. We have, therefore, investigated the requirements for antigen density and costimulatory molecules in direct CTL priming with a prototype cell-based vaccine that uses a signal sequence-containing minigene to direct expression of a tumor-specific CTL epitope to the endoplasmic reticulum. This design limits sources of antigen available to professional APC in the host and, thereby, the contribution of cross-priming. Induction of antitumor CTL immunity by our prototype APC was shown to solely involve direct priming, independent of host APC, NKI.1+ cells, and CD4+ T cell help. CTL induction through this mechanism required the engineered APC to express the B7-1 molecule as well as a sufficiently high density of peptide/MHC complexes at its surface. Our data, in contrast to previous studies using modified tumor cells, clearly define the antigenic and costimulatory requirements for a suitably engineered "artificial" APC to directly prime peptide-specific CTL in vivo, and demonstrate that the signal sequence minigene approach allows the engineering of highly effective and well-defined cellular vaccines for activation of CTL against epitopes of choice.     

The hyperglycemic dehydration syndrome

Two patients, men aged 47 and 64 years, were found in a comatose condition in their homes, after a period of fatigue, polyuria and polydipsia. They had not been known to suffer from diabetes mellitus, but now displayed a hyperglycaemic hyperosmolar non-ketoacidotic disorder as the first manifestation of diabetes mellitus type 2. In that condition, just sufficient insulin is present to counteract ketone production, but not enough to prevent hyperglycaemia. Neurological and thromboembolic manifestations, possibly fatal, may result from severe dehydration brought about by a vicious circle in which osmotic diuresis reduces the effective circulating volume, causing renal function to decrease and hyperglycaemia to increase even more. Both patients recovered fully after adequate treatment with solutions of NaCl and glucose.