Obstacles and limitations of transfer factor biological activity assay design.

Transfer factor (TF) is a heterogeneous mix of low-molecular weight molecules obtained from dialyzed leukocyte extract that is capable of transferring cell-mediated immunity. As an immunostimulatory drug TF is used to improve treatment of infectious diseases, allergies, cancer and immune deficiencies. The main benefit of TF preparations as immunotherapeutic agents is the induction of a rapid immune response and the potential of TF as an adjuvant in combination with other drugs might lead to development of novel approaches to combat various diseases in the future. The process of TF preparation is rather simple. However, with respect to fact that TF is composed by several multifunction molecules, it is likely that during the activity measurement based only on one single parameter, other TF biological activities might be overlooked. In addition, separated TF components might display synergetic activity effect. According to recent European Pharmacopoeia there is no general protocol for immuno-stimulatory drugs (including TF) activity measurement available. Nevertheless, for the process of TF preparation, control of input material and for final pharmaceutical product batches it is inevitable to guaranty proper quality control including appropriate in vivo or in vitro test(s) for TF biological activity assay. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic problem, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. The currently used method for testing biological activity of TF is the in vitro MTT cells proliferation assay that is recognized by control authorities in Slovak Republic. Keywords: immune system; transfer factor; dialysable leukocyte extract; diseases; MTT cells proliferation assay.

High serum carbonic anhydrase IX predicts shorter survival in head and neck cancer.

OBJECTIVES: The objective of the study was to investigate prognostic and predictive value of pretreatment soluble carbonic anhydrase IX (CA IX) blood serum concentration in patients with locally advanced head and neck cancer. BACKGROUND: Increased expression of CA IX in tumor tissues has been associated with treatment resistance and worth prognosis. Soluble form of CA IX, released from tumor cells, is detectable in blood serum and could be a convenient predictive factor of treatment effectiveness that would enable treatment individualization. METHODS: The prospective study evaluated 48 patients with locally advanced squamous cell carcinomas of head and neck, treated by radiotherapy or chemo-radiotherapy. Pretreatment soluble CA IX serum concentrations were examined using enzyme-linked immunosorbent assay. RESULTS: Soluble CA IX serum concentration failed to predict radiotherapy effectiveness in the studied patient population (p = 0.26). However, high CA IX serum concentrations have been associated with shorter overall survival (p = 0.035) CONCLUSION: High pretreatment CA IX serum concentration is a negative prognostic factor in locally advanced head and neck cancer patients (Tab. 1, Fig. 2, Ref. 23).

[Relation between carbonic anhydrase IX serum level, hypoxia and radiation resistance of head and neck cancers]. / Vztah medzi sérovou hladinou karboanhydrázy IX, hypoxiou a rádiorezistenciou nádorov hlavy a krku.

BACKGROUND: Hypoxia of locally advanced head and neck cancers is one of the main causes of their radiation resistance that presents clinically as a persistence of residual tumor disease after radiation therapy. Therefore, detection of tumor hypoxia could be an important predictor of treatment efficacy. Carbonic anhydrase IX (CA IX) is a protein, coded by a homonymous gene, the expression of which increases in tumor tissues at hypoxic conditions. Hence, CA IX represents an endogenic marker of tumor hypoxia, identifiable in tumor tissues, and its soluble extracellular domain can also be detected in body fluids of the patient. The primary endpoint of this study was to explore whether a correlation exists between CA IX serum level and the residual tumor disease after therapy. The secondary endpoint was to find out how the serum concentration of CA IX changes during the course of fractionated radiation therapy. MATERIALS AND METHODS: The presented prospective monocentric clinical study evaluated a population of 30 patients with locally advanced squamous cell head and neck cancers, treated by radiation therapy or concurrent chemo radiation therapy with a curative intent. The serum concentration of the soluble form of CA IX was examined from a venous blood sample, using sandwich enzyme linked immunosorbent assay (ELISA). The blood samples were obtained before the treatment initiation, in the middle of radiation therapy, at the time of finishing radiation therapy and six weeks after the treatment completion. RESULTS: We found a substantial variability in the CA IX levels measured in the examined population, ranging 0- 1,696 pg/ ml. We found no significant changes in the mean value of CA IX concentration during the course of radiation therapy and after the treatment completion. In 11 patients (36.7%), the treatment resulted in complete remission of the disease. In these patients, lower average pretreatment levels of CA IX were noted when compared to patients with persistence of residual tumor disease (37.57 vs 77.47; p = 0.154). CONCLUSION: The results indicate that serum level of CA IX in patients with locally advanced head and neck cancers does not change significantly during the course of fractionated radiation therapy. The relation between CA IX serum level and residual tumor disease after radiation therapy requires verification on a larger population of patients.

Modulation of cell surface density of carbonic anhydrase IX by shedding of the ectodomain and endocytosis.

Carbonic anhydrase IX (CA IX) is a cell surface protein frequently present in human tumors but not in the corresponding normal tissues. Expression of CA IX is primarily determined by the strong transcriptional activation of the gene encoding CA IX by hypoxia via the hypoxia-inducible transcription factor HIF-1. However, the ultimate abundance of the CA IX protein on the cell surface can be affected by additional mechanisms, including ectodomain shedding and endocytosis. In this paper, we summarize the basic knowledge on how these processes modulate CA IX expression at the post-translational level. We propose that the regulation of the CA IX protein residence in the plasma membrane can influence its biological function as well as its clinical exploitation.

The hypoxia-sensor carbonic anhydrase IX affects macrophage metabolism, but is not a suitable biomarker for human cardiovascular disease.

Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD). Transmembrane protein carbonic anhydrase IX (CAIX) is hypoxia-induced and can be shed into the circulation as soluble CAIX (sCAIX). As plaque macrophages are hypoxic, we hypothesized a role for CAIX in macrophage function, and as biomarker of hypoxic plaque burden and CVD. As tumor patients with probable CVD are treated with CAIX inhibitors, this study will shed light on their safety profile. CAIX co-localized with macrophages (CD68) and hypoxia (pimonidazole), and correlated with lipid core size and pro-inflammatory iNOS+ macrophages in unstable human carotid artery plaques. Although elevated pH and reduced lactate levels in culture medium of CAIX knock-out (CAIXko) macrophages confirmed its role as pH-regulator, only spare respiratory capacity of CAIXko macrophages was reduced. Proliferation, apoptosis, lipid uptake and expression of pro- and anti-inflammatory genes were not altered. Plasma sCAIX levels and plaque-resident CAIX were below the detection threshold in 50 and 90% of asymptomatic and symptomatic cases, respectively, while detectable levels did not associate with primary or secondary events, or intraplaque hemorrhage. Initial findings show that CAIX deficiency interferes with macrophage metabolism. Despite a correlation with inflammatory macrophages, plaque-resident and sCAIX expression levels are too low to serve as biomarkers of future CVD.

Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2.

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.

Monoclonal antibodies to the distinct antigenic sites on glycoproteins C and B and their protective abilities in herpes simplex virus infection.

The relative importance of the humoral immune response to various antigenic sites on the glycoproteins C and B (gC, gB) of herpes simplex virus (HSV) was evaluated in BALB/c and DBA/2 mice passively immunized with monoclonal antibodies (MoAbs) and then challenged with lethal dose of infectious virus. Eight MoAbs to three topographically distinct antigenic sites on gC and eight MoAbs to two distinct antigenic sites on gB were selected. The results indicated that any antigenic site on gC and gB contains epitopes for the protective immunity. However, individual MoAbs to different epitopes of the same antigenic site (i.e. antigenic site III on gC, and antigenic site II on gB) varied extremely in their protective ability. The protection did not correlate with the virus neutralization in vitro whereas it correlated significantly with the immune cytolysis in the presence of complement. The information about protective epitopes is essential for understanding the immunology of HSV infection at molecular level and may have implications for the design of HSV vaccine.

Monoclonal antibodies suitable for type-specific identification of herpes simplex viruses by a rapid culture assay.

Eight monoclonal antibodies (MAbs) to herpes simplex viruses 1 and/or 2 (HSV-1, HSV-2) were tested for their reactivity with 190 previously typed HSV-positive clinical specimens in order to determine their suitability for use as type-specific reagents. Using a rapid culture assay we found that two MAbs (T51 and T96) identified HSV-1 in all the 94 specimens, previously found HSV-1-positive, but did not react with the 96 specimens, previously found HSV-2-positive. In contrast, one MAb (303) confirmed the presence of HSV-2 in all the specimens, previously found HSV-2-positive, but did not react with any of the 94 specimens, previously found HSV-1-positive. These three type-specific MAbs allow for a rapid type-specific identification of HSV in infected cultures. One type-common MAb (T111) reacted with all HSV-positive cultures. This MAb can be used as an excellent reagent for detection of HSV infection.

Sequence and characterisation of the Z gene encoding ring finger protein of the lymphocytic choriomeningitis virus MX strain.

We have cloned and characterised a cDNA encoding Z protein of recently identified MX strain of lymphocytic choriomeningitis virus (LCMV) persistently infecting human MaTu cells. Deduced amino acid sequence of LCMV MX Z protein showed 88.9% identity with that of the LCMV Armstrong (ARM) strain and 80.9% identity with that of the LCMV Traub (TRA) strain. It contained conserved zinc-binding RING finger domain and C-terminal proline-rich region. Northern blot analysis of total RNA from MaTu cells revealed presence of abundant truncated forms of L RNA. Z protein-specific rabbit antibodies were produced to glutathione S-transferase (GST)-Z fusion protein expressed in E. coli and used for the detection of Z protein in MaTu cells. Western blot and immunofluorescence analyses detected relatively high levels of Z protein indicating its role in maintenance of persistent LCMV.

Carbonic anhydrase IX as an anticancer therapy target: preclinical evaluation of internalizing monoclonal antibody directed to catalytic domain.

Carbonic anhydrase IX (CA IX) is a suitable target for various anticancer strategies. It is a cell surface protein that is present in human tumors, but not in the corresponding normal tissues. Expression of CA IX is induced by hypoxia and correlates with cancer prognosis in many tumor types. Moreover, CA IX is functionally implicated in cancer progression as a pro-survival factor protecting cancer cells against hypoxia and acidosis via its capability to regulate pH and cell adhesion. Cancer-related distribution of CA IX allows for targeting cancer cells by antibodies binding to its extracellular domain, whereas functional involvement of CA IX opens the possibility to hit cancer cells by blocking their adaptation to physiologic stresses via inhibition of CA IX enzyme activity. The latter strategy is recently receiving considerable attention and great efforts are made to produce CA IX-selective inhibitor derivatives with anticancer effects. On the other hand, targeting CA IX-expressing cells by immunotherapy has reached clinical trials and is close to application in treatment of renal cell carcinoma patients. Nevertheless, development and characterization of new CA IX-specific antibodies is still ongoing. Here we describe a mouse monoclonal antibody VII/20 directed to catalytic domain of CA IX. We show that upon binding to CA IX, the VII/20 MAb undergoes efficient receptor-mediated internalization, which is a process regulating abundance and signaling of cell surface proteins and has considerable impact on immunotherapy. We evaluated biological properties of the MAb and demonstrated its capacity to elicit anti-cancer effect in mouse xenograft model of colorectal carcinoma. Thus, the VII/20 MAb might serve as a tool for preclinical studies of immunotherapeutic strategies against non-RCC tumors. These have not been explored so far and include broad spectrum of cancer types, treatment of which might benefit from CA IX-mediated targeting.

Cancer-associated carbonic anhydrases and their inhibition.

Cells of the growing tumor tissue are exposed to physiological stresses connected with insufficient delivery of oxygen (hypoxia) and accumulation of acidic products of the glycolytic metabolism (acidosis). Adaptation to these microenvironmental stresses involves remodeling of the cellular expression program mediated by hypoxia-inducible factor (HIF), which activates broad array of genes functionally involved in angiogenesis, anaerobic glycolysis, de-adhesion, invasion etc. This leads to increased aggressiveness of tumors, metastatic spread and poor response to therapy. Genes coding for transmembrane carbonic anhydrase (CA) isoforms IX and XII are induced in response to low oxygen as a part of the hypoxic transcriptome. Moreover, CA IX is a direct target of HIF and serves as a surrogate marker of hypoxia and prognostic indicator. Its expression is strongly linked to different types of tumors with the HIF pathway activated due to genetic defect or physiological hypoxia. CA IX (and possibly also CA XII) is participates in pH regulation, which is important for survival of hypoxic cells. Both enzymes are therefore promising therapeutic molecules targetable by inhibitors of CA activity. Some of these sulfonamide compounds and their derivatives are capable to block CA-mediated pH regulation in hypoxia. This review summarizes research data related to distribution, regulation and functional aspects of CA IX and CA XII, and describes emerging possibilities for clinical exploitation of CA inhibitors as imaging tools and anticancer drugs.

Alternative splicing variant of the hypoxia marker carbonic anhydrase IX expressed independently of hypoxia and tumour phenotype.

CA IX is a hypoxia-induced, cancer-associated carbonic anhydrase isoform with functional involvement in pH control and cell adhesion. Here we describe an alternative splicing variant of the CA9 mRNA, which does not contain exons 8-9 and is expressed in tumour cells independently of hypoxia. It is also detectable in normal tissues in the absence of the full-length transcript and can therefore produce false-positive data in prognostic studies based on the detection of the hypoxia- and cancer-related CA9 expression. The splicing variant encodes a truncated CA IX protein lacking the C-terminal part of the catalytic domain. It shows diminished catalytic activity and is intracellular or secreted. When overexpressed, it reduces the capacity of the full-length CA IX protein to acidify extracellular pH of hypoxic cells and to bind carbonic anhydrase inhibitor. HeLa cells transfected with the splicing variant cDNA generate spheroids that do not form compact cores, suggesting that they fail to adapt to hypoxic stress. Our data indicate that the splicing variant can functionally interfere with the full-length CA IX. This might be relevant particularly under conditions of mild hypoxia, when the cells do not suffer from severe acidosis and do not need excessive pH control.

Modulation by 6-hydroxydopamine of expression of the phenylethanolamine N-methyltransferase (PNMT) gene in the rat heart during immobilization stress.

Phenylethanolamine N-methyltransferase (PNMT) is the final enzyme in the catecholamine synthesizing cascade that converts noradrenaline (NA) to adrenaline (Adr). Both of these catecholamines are physiologically important hormones and neurotransmitters in mammals with profound influence on the activity of the cardiovascular system. Although PNMT activity and gene expression have been reported in the neonatal and also adult rat heart, little is known about the identity of the cells expressing PNMT mRNA. In this study, we have shown that besides PNMT in neuronal and intrinsic cardiac cells, this enzyme is expressed also in rat cardiomyocytes, as shown by immunofluorescence in isolated cardiomyocytes. To determine which cells in the heart more sensitively show stress-induced changes in PNMT mRNA expression, we performed chemical sympathectomy by administration of 6-hydroxydopamine (6-OHDA), which destroys catecholaminergic terminals. We determined PNMT mRNA levels in the left atria and ventricles of control and stressed rats. In the rats treated with 6-OHDA, PNMT mRNA levels were not changed under normal, physiological conditions compared to vehicle treated rats. Similar results were observed on isolated cardiomyocytes from control and 6-OHDA treated rats. However, 6-OHDA treatment prevented immobilization-induced increase in PNMT mRNA expression. The results allow us to propose that in the heart, the immobilization-induced increase in PNMT gene expression is probably not in cardiomyocytes, but in neuronal cells.

Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17.

Carbonic anhydrase IX (CA IX) is a transmembrane protein whose expression is strongly induced by hypoxia in a broad spectrum of human tumours. It is a highly active enzyme functionally involved in both pH control and cell adhesion. Its presence in tumours usually indicates poor prognosis. Ectodomain of CA IX is detectable in the culture medium and body fluids of cancer patients, but the mechanism of its shedding has not been thoroughly investigated. Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels. Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFalpha-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent. Our results suggest that the cleavage of CA IX ectodomain is a regulated process that responds to physiological factors and signal transduction stimuli and may therefore contribute to adaptive changes in the protein composition of tumour cells and their microenvironment.