Distinct responses between healthy and cirrhotic human livers upon lipopolysaccharide challenge: possible implications for acute-on-chronic liver failure.

Patients with acute-on-chronic liver failure (ACLF) are at risk of developing acute hepatic decompensation and organ failures with an unraveled complex mechanism. An altered immune response toward insults in cirrhotic compared with healthy livers may contribute to the ACLF development. Therefore, we aim to investigate the differences in inflammatory responses between cirrhotic and healthy livers using human precision-cut liver slices (PCLSs) upon the lipopolysaccharide (LPS) challenge. PCLSs prepared from livers of patients with cirrhosis or healthy donors of liver transplantation were incubated ex vivo with or without LPS for up to 48 h. Viability test, qRT-PCR, and multiplex cytokine assay were performed. Regulation of the LPS receptors during incubation or with LPS challenge differed between healthy versus cirrhotic PCLSs. LPS upregulated TLR-2 in healthy PCLSs solely (P < 0.01). Culturing for 48 h induced a stronger inflammatory response in the cirrhotic than healthy PCLS. Upon LPS stimulation, cirrhotic PCLSs secreted more proinflammatory cytokines (IL-8, IL-6, TNF-α, eotaxin, and VEGF) significantly and less anti-inflammatory cytokine (IL-1ra) than those of healthy. In summary, cirrhotic PCLSs released more proinflammatory and less anti-inflammatory cytokines after LPS stimuli than healthy, leading to dysregulated inflammatory response. These events could possibly resemble the liver immune response in ACLF.NEW & NOTEWORTHY Precision-cut liver slices (PCLSs) model provides a unique platform to investigate the different immune responses of healthy versus cirrhotic livers in humans. Our data show that cirrhotic PCLSs exhibit excessive inflammatory response accompanied by a lower anti-inflammatory cytokine release in response to LPS; a better understanding of this alteration may guide the novel therapeutic approaches to mitigate the excessive inflammation during the onset of acute-on-chronic liver failure.

Tumor-infiltrating CD14-positive myeloid cells and CD8-positive T-cells prolong survival in patients with cervical carcinoma.

One of the hallmarks of cancer is the influx of myeloid cells. In our study, we investigated the constitution of tumor-infiltrating myeloid cells and their relationship to other tumor-infiltrating immune cells, tumor characteristics and the disease-specific survival of patients with cervical cancer (CxCa). Triple-color immunofluorescence confocal microscopy was used to locate, identify and quantify macrophages (CD14), their maturation status (CD33) and their polarization (CD163) in a cohort of 86 patients with cervical carcinoma. Quantification of the numbers of myeloid cells revealed that a strong intraepithelial infiltration of CD14+ cells, and more specifically the population of CD14+CD33-CD163- matured M1 macrophages, is associated with a large influx of intraepithelial T lymphocytes (p = 0.008), improved disease-specific survival (p = 0.007) and forms an independent prognostic factor for survival (p = 0.033). The intraepithelial CD8+ T-cell and regulatory T-cell (Treg) ratio also forms an independent prognostic factor (p = 0.010) and combination of these two factors reveals a further increased benefit in survival for patients whose tumor displays a dense infiltration with intraepithelial matured M1 macrophages and a high CD8 T-cell/Treg ratio, indicating that both populations of immune cells simultaneously improve survival. Subsequently, we made a heatmap including all known immune parameters for these patients, whereby we were able to identify different immune signatures in CxCa. These results indicate that reinforcement and activation of the intratumoral M1 macrophages may form an attractive immunotherapeutic option in CxCa.

Role of IL-12p40 in cervical carcinoma.

BACKGROUND: Previously, we have shown that low IL-12p40 mRNA expression by cervical cancer cells is associated with a poor survival of cervical cancer patients. As IL-12p40 is both a subcomponent of interleukin (IL)-12 and IL-23, the aim of this study was to elucidate the role of IL-12p40 in cervical cancer. METHODS: We have measured the expression of IL-23p19 mRNA, IL-12p35 mRNA and IL-12p40 mRNA using mRNA in situ hybridisation. The IL-1 and IL-6 were measured by immunohistochemistry. RESULTS: As IL-23 is a component of the IL-17/IL-23 pathway, a pathway induced by IL-1 and IL-6 in humans, we have studied IL-1 and IL-6 expression. Only a high number of stromal IL-6-positive cells was shown to associate with poor disease-specific survival. The worst disease-specific survival was associated with a subgroup of patients that displayed a high number of IL-6-positive cells and low IL-12p40 expression (P<0.001). Both a high number of IL-6-positive cells and a high number of IL-6-positive cells, plus low IL-12p40 expression were shown to be clinicopathological parameters independent of lymph node metastasis, parametrial involvement and Sedlis score (P=0.009 and P=0.007, respectively). CONCLUSION: Our results with IL-6 and IL-12p40 are in accordance with the hypothesis that the IL-17/IL-23 pathway has a suppressive role in cervical cancer.

CXCR7 expression is associated with disease-free and disease-specific survival in cervical cancer patients.

BACKGROUND: The CXC chemokine receptor (CXCR)7 is involved in tumour development and metastases formation. The aim of the present study was to determine protein expression of CXCR7, its putative co-receptors epidermal growth factor receptor (EGFR) and CXCR4, its predominant ligand CXCL12, their co-dependency and their association with survival in cervical cancer patients. METHODS: CXC chemokine receptor 7, EGFR, CXCR4 and CXCL12 expression were determined immunohistochemically in 103 paraffin-embedded, cervical cancers. Subsequently, associations with patient characteristics were assessed and survival analyses were performed. RESULTS: CXC chemokine receptor 7 was expressed by 43% of tumour specimens, in a large majority of cases together with either EGFR or CXCR4 (double positive), or both (triple positive). The CXCR7 expression was associated with tumour size (P=0.013), lymph node metastasis (P=0.001) and EGFR expression (P=0.009). CXC chemokine receptor 7 was independently associated with disease-free survival (hazard ratio (HR)=4.3, 95% confidence intervals (CI) 1.7-11.0, P=0.002), and strongly associated with disease-specific survival (HR=3.9, 95% CI 1.5-10.2, P=0.005). CONCLUSION: CXC chemokine receptor 7 expression predicts poor disease-free and disease-specific survival in cervical cancer patients, and might be a promising new therapeutic marker. In a large majority of cases, CXCR7 is co-expressed with CXCR4 and/or EGFR, supporting the hypothesis that these receptors assist in CXCR7 signal transduction.

Systemic and local human papillomavirus 16-specific T-cell immunity in patients with head and neck cancer.

Squamous cell carcinomas of the head and neck (HNSCC), in particular those of the oropharynx, can be caused by human papilloma virus Type 16 (HPV16). Whereas these HPV-induced oropharyngeal carcinomas may express the HPV16 E6 and E7 oncoproteins and are associated with better survival, the nonvirally induced HNSCC are associated with overexpression of p53. In this study we assessed the presence of systemic and local T cells reactive against these oncoproteins in HNSCC. An exploratory study on the presence, type and function of HPV16- and/or p53-specific T cells in the blood, tumor and/or metastatic lymph node as measured by several immune assays was performed in an unselected group of 50 patients with HNSCC. Tumor tissue was tested for HPV DNA and the overexpression of p53 protein. Almost all HPV16+ tumors were located in the oropharynx. Circulating HPV16- and p53-specific T cells were found in 17/47 and 7/45 tested patients. T cells were isolated from tumor cultures and/or lymph nodes of 20 patients. HPV16-specific T cells were detected in six of eight HPV+ tumors, but in none of the 12 HPV-tumors. Tumor-infiltrating p53-specific T cells were not detected. In depth analysis of the HPV16-specific T-cell response revealed that this response comprised a broad repertoire of CD4+ T-helper Type 1 and 2 cells, CD4+ regulatory T cells and CD8+ T cells reactive to HPV16. The local presence of HPV16-specific T-cell immunity in HPV16-induced HNSCC implicates a role in the antitumor response and support the development of immunotherapy for HNSCC.

Critical role of endoglin in tumor cell plasticity of Ewing sarcoma and melanoma.

Tumor cell plasticity enables certain types of highly malignant tumor cells to dedifferentiate and engage a plastic multipotent embryonic-like phenotype, which enables them to 'adapt' during tumor progression and escape conventional therapeutic strategies. This plastic phenotype of aggressive cancer cells enables them to express endothelial cell-specific markers and form tube-like structures, a phenotype that has been linked to aggressive behavior and poor prognosis. We demonstrate here that the transforming growth factor (TGF)-ß co-receptor endoglin, an endothelial cell marker, is expressed by tumor cells and its expression correlates with tumor cell plasticity in two types of human cancer, Ewing sarcoma and melanoma. Moreover, endoglin expression was significantly associated with worse survival of Ewing sarcoma patients. Endoglin knockdown in tumor cells interferes with tumor cell plasticity and reduces invasiveness and anchorage-independent growth in vitro. Ewing sarcoma and melanoma cells with reduced endoglin levels showed reduced tumor growth in vivo. Mechanistically, we provide evidence that endoglin, while interfering with TGF-ß signaling, is required for efficient bone morphogenetic protein, integrin, focal adhesion kinase and phosphoinositide-3-kinase signaling in order to maintain tumor cell plasticity. The present study delineates an important role of endoglin in tumor cell plasticity and progression of aggressive tumors.

Expression of endoglin (CD105) in cervical cancer.

In this study, we have investigated the role of endoglin (CD105), a regulator of transforming growth factor (TGF)-beta(1) signalling on endothelial cells, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) in cervical cancer. We have measured the number and determined the location of both newly formed (CD105-positive) and the overall number of (CD31-positive) blood vessels, and bFGF and VEGF-A expression using immunohistochemistry in 30 cervical carcinoma specimens. Vascular endothelial growth factor-A mRNA expression was determined using RNA-in situ hybridisation. CD105- and CD31-positive vessels and bFGF- and VEGF-A-positive cells were predominantly present in the stroma. The presence of CD105- and CD31-positive vessels in the stroma did neither correlate with the number of VEGF-A-positive cells nor the number of bFGF-positive cells. However, the number of CD105- and CD31-positive vessels was associated with the expression of VEGF-A mRNA in the epithelial cell clusters (P=0.013 and P=0.005, respectively). The presence of CD105-positive and CD31-positive vessels was associated with the expression of alphavbeta6 (a TGF-beta(1) activator; P=0.013 and P=0.006, respectively). Clinically, the number of CD105-positive vessels associated with the number of lymph node metastasis (P<0.001). Furthermore, the presence of CD105-positive vessels within the epithelial cell clusters associated with poor disease-free survival (P=0.007).

Elevated expression of SerpinA1 and SerpinA3 in HLA-positive cervical carcinoma.

In cervical cancer, an important mechanism by which tumour cells escape immune surveillance is loss of HLA class I, enabling tumours to evade recognition and lysis by cytotoxic T lymphocytes. Some tumours, however, escape from immune surveillance without accumulating defects in antigen presentation. We hypothesized that tumours with no or partial loss of HLA class I develop alternative mechanisms to prevent immune elimination. To investigate this hypothesis, genome-wide expression profiling using Illumina arrays was performed on cervical squamous cell carcinomas showing overall loss of HLA class I, partial, and normal HLA class I protein expression. Statistical analyses revealed no significant differences in gene expression between tumours with partial (n = 11) and normal HLA class I expression (n = 10). Comparison of tumours with normal/partial HLA class I expression (n = 21) with those with overall loss of HLA class I expression (n = 11) identified 150 differentially expressed genes. Most of these genes were involved in the defence response (n = 27) and, in particular, inflammatory and acute phase responses. Especially SerpinA1 and SerpinA3 were found to be up-regulated in HLA-positive tumours (3.6- and 8.2-fold, respectively), and this was confirmed by real-time PCR and immunohistochemistry. In a group of 117 tumours, high SerpinA1 and SerpinA3 expression in association with normal/partial HLA expression correlated significantly with poor overall survival (p = 0.035 and p = 0.05, respectively). Thus, HLA-positive tumours are characterized by higher expression of genes associated with an inflammatory profile. In addition, expression of the acute phase proteins SerpinA1 and SerpinA3 in HLA-positive tumours is associated with worse prognosis.

Overexpression of the alpha v beta 6 integrin in cervical squamous cell carcinoma is a prognostic factor for decreased survival.

Cervical squamous cell carcinomas are composed histologically of tumour cell islands surrounded by varying amounts of tumour stroma, the amount and composition of which are influenced by local TGF-beta(1). TGF-beta(1) is secreted in an inactive complex with latency-associated peptide (LAP). Both LAP and the extracellular matrix (ECM) protein fibronectin are important ligands for the integrin receptor alpha v beta 6. While alpha v beta 6 is only weakly expressed by normal epithelia, it is up-regulated in different carcinomas where it generally reflects a more aggressive phenotype. In cervical cancer, the expression of alpha v beta 6 has not thus far been investigated. Given the ability of alpha v beta 6 both to activate TGF-beta(1) and to interact with fibronectin, we studied correlations between the expression of these components and disease parameters in a large cohort of cervical cancer specimens. We analysed alpha v beta 6 expression using immunohistochemistry in primary cervical squamous carcinomas of FIGO stage IA to IIB patients and correlated the findings with formerly investigated fibronectin and TGF-beta(1) expression and clinico-pathological parameters. alpha v beta 6 expression was also examined in cervical intra-epithelial neoplasia (CIN) and lymph node metastases. alpha v beta 6 was only weakly expressed in normal epithelium but clearly up-regulated in CIN lesions. In carcinomas, strong expression of alpha v beta 6 in tumour cells correlated with different clinico-pathological parameters and with worse overall and disease-free survival. Furthermore, alpha v beta 6 expression correlated positively with TGF-beta(1) mRNA expression as well as with fibronectin expression. Overexpression of alpha v beta 6 in cervical squamous carcinomas is an unfavourable prognostic factor. This might reflect an increased capacity of alpha v beta 6-expressing tumour cells to migrate in a fibronectin-rich ECM and/or to activate TGF-beta(1) at the tumour/stroma interface, both of which processes may contribute to cervical cancer progression.

Chlamydia trachomatis infections in Nicaragua: preliminary results from a competitive voucher scheme to prevent and treat sexually transmitted infections and HIV/AIDS among sex workers.

A donor-supported competitive voucher scheme in Nicaragua provides prevention and treatment services for sexually transmitted infections (STIs) and HIV/AIDS to high-risk populations such as sex workers and their partners and clients. Beyond detecting and treating STIs, HIV and AIDS, these health services can also raise awareness of risks and promote safer behavior, leading to widespread benefits. This review describes the voucher scheme, summarizes published data on Chlamydia trachomatis infection in Nicaragua and provides preliminary prevalence data on C. trachomatis obtained in 2003 through the voucher scheme.

The absence of CCL2 expression in cervical carcinoma is associated with increased survival and loss of heterozygosity at 17q11.2.

To determine whether CCL2 mRNA expression is beneficial or detrimental for cervical cancer patients, the association between the expression of this molecule by cervical tumour cells, the number of tumour-associated macrophages, and clinicopathological parameters such as recurrence, relapse-free survival, and overall patient survival was investigated. In cervical cancer samples from 93 untreated cervical cancer patients, the CCL2 mRNA expression level was quantified using RNA in situ hybridization and verified using real-time quantitative RT-PCR. The number of tumour-associated macrophages was determined using immunohistochemistry. Furthermore, the study investigated whether lack of CCL2 expression was due to genetic alterations near the 17q11.2 (CCL2 genomic) region. CCL2 mRNA expression by cervical tumour cells was associated with the number of tumour-associated macrophages (p < 0.001). Lack of CCL2 mRNA expression (15 samples; 16%) was associated with increased cumulative relapse-free survival (log rank test, p = 0.030), increased cumulative overall survival (log rank test, p = 0.024), less post-operative surgery, reduced local and distant recurrence, reduced vascular invasion, and smaller tumour size (<40 mm). The absence of CCL2 mRNA expression corresponded with loss of heterozygosity (LOH) at 17q11.2 in five of six samples. The increased cumulative relapse-free survival and cumulative overall survival of cervical cancer patients lacking tumour cell-associated CCL2 mRNA suggest that the tumour-associated macrophages support tumour progression, presumably by promoting angiogenesis and production of growth factors.

CD55 expression patterns on intestinal neuronal tissue are divergent from the brain.

BACKGROUND: and objectives: In this study, we investigated how enteric plexuses protect themselves from complement mediated attack. For this purpose, the expression patterns of membrane bound complement regulatory proteins (mCRP) and their association with C3 deposition was determined. In addition, mCRP expression patterns of enteric plexuses were compared with those in the central nervous system (CNS). METHODS: Immunohistochemical stainings were performed to discriminate neuronal cells from glial cells and to detect the presence of CD46, CD55, CD59, and C3d. RNA in situ hybridisation (RISH) was used to determine the cell types that produce CD55 mRNA. RESULTS: Enteric plexuses minimally expressed CD46 whereas CD55 and CD59 were highly expressed. CD55 expression was also observed in a ring around Auerbach's plexuses which was not observed for CD46 and CD59. C3d was deposited around the plexuses but plexus cells themselves did not stain for C3d. In contrast with CNS neurones, enteric neurones were shown to express CD55 whereas enteric glial cells did not. This was confirmed with CD55 RISH. Phospholipase C mediated cleavage of CD55 demonstrated that CD55 was most likely attached to elastic fibres surrounding the plexus. Attached CD55 might protect CD55 negative glial cells from complement mediated injury during inflammatory reactions. CD55 on elastic fibres surrounding the plexuses most likely originated from enteric neuronal cells. CONCLUSION: In contrast with the CNS, enteric neurones express CD55 and enteric glial cells lack CD55 expression. CD55, produced by neuronal cells, attached to elastic fibres surrounding the plexuses is proposed to protect the CD55 negative glial cells within plexuses.

Membrane-bound complement regulatory proteins inhibit complement activation by an immunotherapeutic mAb in a syngeneic rat colorectal cancer model.

MAb-mediated immunotherapy offers a potential tool for destroying metastasizing colorectal tumor cells. Promising results have been obtained by using xenograft models. However, overexpression of membrane-bound complement regulatory proteins (mCRP) impedes complement-mediated destruction of tumor cells in vitro. mCRP operate in a species selective manner. Therefore a syngeneic animal model is needed to investigate the contribution of mCRP in mAb-mediated immunotherapy. Here we present a syngeneic rat colorectal carcinoma model, which fulfills the conditions necessary to investigate the effect of mCRP expression on mAb-mediated immunotherapy of metastases of solid tumors.CC531 rat colorectal cancer cells were injected subcapsularly into the liver of syngeneic WAG/Rij rats. Four mAb (MG1(IgG2a), MG2(IgG2a), MG3(IgG3) and MG4(2a)(IgG2a)) directed against CC531 cells, were tested for their complement activating abilities in vitro and tumor homing capacities in vivo. Only MG4(2a) was found to activate complement in vitro and home to the tumor cells in vivo. This mAb induced C3-deposition and C-mediated lysis of CC531 cells in vitro when the effects of the C-inhibitors Crry/p65 and CD59 were neutralized. This implies an important role for these mCRP in restricting the effector functions of tumor-associated mAb on these cells. Although C activation could be induced by MG4(2a) in situ on tumor tissue sections, no deposition of C3 could be found on the tumor cells positive for MG4(2a) in vivo. This suggests that complement activation in vivo was inhibited by mCRP. The results indicate the suitability of this syngeneic animal model for studying the effects of mAb immunotherapy. However, the effect of mCRP on tumor cells need to be overcome, e.g. by the use of mAb against tumor antigens and mCRP.

Cytokines affect resistance of human renal tumour cells to complement-mediated injury.

Overexpression of membrane-bound complement regulatory proteins (mCRPs) on tumour cells may hamper the effect of immunotherapy with complement-activating monoclonal antibody (MoAb). Therefore, it is important to investigate whether cytokines can downregulate the expression of mCRP on tumour cells. In this study, the effect of 10 cytokines on the expression of the mCRP CD46, CD55 and CD59 and the renal tumour-associated antigen G250/MN/CAIX on four human renal tumour cell lines and proximal tubular epithelial cells was determined by flow cytometry. In addition, it was measured whether changes in the expression of the classical pathway regulatory proteins CD55 and CD59 had an effect on C3 deposition and lysis. Interleukin-1beta (IL-1beta) consistently downregulated the expression of CD46 and CD59; IL-4 consistently downregulated the expression of CD46 and transforming growth factor-beta1, consistently downregulated the expression of both CD46 and CD55. However, treatment with IL-1beta and IL-4 also decreased the expression of G250/MN/CAIX. Changes in the expression of CD55 and CD59 were associated with changes in the amount of C3 deposited and the extent of complement-mediated lysis, respectively. This suggests that clinical immunotherapy, consisting of treatment with cytokines and MoAb, may induce either up- or downregulation of CD55 or CD59 and thus affect the effectiveness of immunotherapy with MoAb.

Pharmacokinetics of differently designed immunoliposome formulations in rats with or without hepatic colon cancer metastases.

PURPOSE: Compare pharmacokinetics of tumor-directed immunoliposomes in healthy and tumor-bearing rats (hepatic colon cancer metastases). METHODS: A tumor cell-specific monoclonal antibody was attached to polyethyleneglycol-stabilized liposomes, either in a random orientation via a lipid anchor (MPB-PEG-liposomes) or uniformly oriented at the distal end of the PEG chains (Hz-PEG-liposomes). Pharmacokinetics and tissue distribution were determined using [3H]cholesteryloleylether or bilayer-anchored 5-fluoro[3H]deoxyuridine-dipalmitate ([3H]FUdR-dP) as a marker. RESULTS: In healthy animals clearance of PEG-(immuno)liposomes was almost log-linear and only slightly affected by antibody attachment; in tumor-bearing animals all liposomes displayed biphasic clearance. In normal and tumor animals blood elimination increased with increasing antibody density; particularly for the Hz-PEG-liposomes, and was accompanied by increased hepatic uptake, probably due to increased numbers of macrophages induced by tumor growth. The presence of antibodies on the liposomes enhanced tumor accumulation: uptake per gram tumor tissue (2-4% of dose) was similar to that of liver. Remarkably, this applied to tumor-specific and irrelevant antibody. Increased immunoliposome uptake by trypsin-treated Kupffer cells implicated involvement of high-affinity Fc-receptors on activated macrophages. CONCLUSIONS: Tumor growth and immunoliposome characteristics (antibody density and orientation) determine immunoliposome pharmacokinetics. Although with a long-circulating immunoliposome formulation, efficiently retaining the prodrug FUdR-dP, we achieved enhanced uptake by hepatic metastases, this was probably not mediated by specific interaction with the tumor cells, but rather by tumor-associated macrophages.

Cytokine profile of cervical cancer cells.

OBJECTIVE: In patients with cervical carcinoma, the presence of cytokines produced by T(H)2 cells, and the presence of an eosinophilic inflammatory infiltrate, has been associated with a less effective immune response and tumor progression. In the present study, we have investigated the cytokine profile of cervical carcinoma cells. In addition, we have measured whether differences in cytokine profiles between normal and malignant cervical epithelial cells are present. METHODS: For this purpose we have determined the mRNA expression patterns of 20 relevant cytokines by RT-PCR and Southern blotting in 3 normal primary cervical epithelial cell cultures (NPE) and 10 cervical cancer cell lines (CCCL). RESULTS: TGF-beta(1), IL-4, IL-12p35, and IL-15 were produced by all CCCL and NPE. TNF-alpha, IL-10, IL-5, and RANTES were present in most NPE, but not in any of the CCCL. MCP-1 was expressed in all CCCL but in only one NPE. The presence of the anti-inflammatory cytokine TGF-beta(1) in cervical carcinomas was confirmed by RNA in situ hybridization on tissue sections of carcinomas from which the CCCL originated. CONCLUSIONS: Our results suggest that cervical carcinoma cells produce immunomodulatory cytokines and that cytokine expression patterns change after malignant transformation. The implications of locally produced cytokines by cervical cancer cells are further discussed.

Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors.

The concept for cellular immunotherapy of solid tumors relies heavily on the capacity of class I MHC-restricted cytotoxic T lymphocytes (CTLs) to eliminate tumor cells. However, tumors often have managed to escape from the cytolytic machinery of these effector cells. Therefore, it is very important to chart the mechanisms through which this escape can occur. Target-cell killing by CTLs involves the induction of apoptosis by two major mechanisms: through death receptors and the perforin/granzyme B (GrB) pathway. Whereas tumors previously were shown to exhibit mechanisms for blocking the death receptor pathway, we now demonstrate that they also can resist CTL-mediated killing through interference with the perforin/GrB pathway. This escape mechanism involves expression of the serine protease inhibitor PI-9/SPI-6, which inactivates the apoptotic effector molecule GrB. Expression of PI-9 was observed in a variety of human and murine tumors. Moreover, we show that, indeed, expression results in the resistance of tumor cells to CTL-mediated killing both in vitro and in vivo. Our data reveal that PI-9/SPI-6 is an important parameter determining the success of T cell-based immunotherapeutic modalities against cancer.

The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds.

Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma have been reported to be functionally monovalent. In a previous paper, we showed that the apparent monovalency of circulating IgG4 antibodies is caused by asymmetry of plasma IgG4-a large fraction has two antigen-binding sites resulting in bispecificity. We postulated that the generation of bispecific antibodies was caused by a post-secretion mechanism, involving the exchange of IgG4 half-molecules (i.e. one heavy and one light chain). This hypothesis was based on the observed instability of the inter-heavy chain disulfide bonds of IgG4. To investigate this instability, we constructed IgG4 mutants and analyzed the covalent interaction between the heavy chains by sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The mutation to serine of one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, resulted in a more stable rather than a more labile inter-heavy chain linkage. Moreover, we confirmed that mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent interaction between the heavy-chains. These two observations suggested an explanation for the observed instability of the inter-heavy chain disulfide bonds: the formation of an alternative, intra-chain cystine. Obviously, this intra-chain cystine cannot be formed in the mutant where Cys226 is replaced by Ser, and cannot easily be formed in the mutant with the IgG1 hinge sequence (Cys-Pro-Pro-Cys) due to the restricted torsional freedom of prolines. We, therefore, postulate that the lack of a covalent heavy-chain interaction in a subpopulation of IgG4 reflects an equilibrium between inter- and intra-chain cystines. Based upon the published structure of the IgG4-related hinge-deleted IgG1 myeloma protein Mcg, we propose a model for the two forms of IgG4 and for the half-molecule exchange reaction, which might result in the formation of bispecific IgG4 antibodies.

Role of NK cells in adoptive immunotherapy of metastatic colorectal cancer in a syngeneic rat model.

This article reviews our immunotherapy research with natural killer (NK) cells in a syngeneic rat colorectal cancer liver and lung metastasis model. Using adoptive transfer of interleukin (IL)-2-activated NK cells, NK cells were shown to selectively infiltrate the tumors. More NK cells were found in tumors when the NK cells were directly injected into tumor-draining blood vessels than when the cells were injected in systemic blood vessels. Under optimal conditions, a limited, though significant, effect of adoptively transferred NK cells on tumor growth was shown. We observed that both endogenous and adoptively transferred NK cells were predominantly present in the stroma surrounding the tumor cell nodules. It is possible that they did not penetrate the nodules containing the tumor cells because of the presence of a basal membrane-like structure around these nodules. Adoptively transferred NK cells may initiate elimination of tumor cells by activating other effector cells, whereas some may eliminate tumor cells by direct cell-cell contact. A diverse array of molecules was shown to be involved in this process. CD45 on NK cells was found to be important in initiating the lysis-inhibitory signal upon binding of 'self' major histocompatibility complex (MHC) class I on potential target cells. Our results indicate that NK-cell cancer therapy is still promising and needs improvement.

Stimulation of bacterial adherence by neutrophil defensins varies among bacterial species but not among host cell types.

Adherence of Haemophilus influenzae to bronchial epithelial cells is enhanced by neutrophil defensins, which are released from activated neutrophils during inflammation [Gorter et al. (1998) J. Infect. Dis. 178, 1067-1078]. In this study, we showed that the adherence of H. influenzae to various epithelial, fibroblast-like and endothelial cell types was significantly enhanced by defensins (20 microg ml(-1)). Defensins stimulated also the adherence of Moraxella catarrhalis, Neisseria meningitidis and nonencapsulated Streptococcus pneumoniae to the NCI-H292 cell line. In contrast, defensins did not affect the adherence of Pseudomonas aeruginosa, encapsulated S. pneumoniae, Escherichia coli and Staphylococcus epidermidis. These results suggest that the defensin-enhanced adherence might support the adherence and possibly persistence of the selected bacterial species using the respiratory tract as port of entry.