Immunization with a circumsporozoite epitope fused to Bordetella pertussis adenylate cyclase in conjunction with cytotoxic T-lymphocyte-associated antigen 4 blockade confers protection against Plasmodium berghei liver-stage malaria

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8+ T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8+ T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-gamma) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.

Effect of periodontal treatment on the serum antibody levels to heat shock proteins

We have shown previously that both humoral and cellular immune responses to heat shock protein 60 (HSP60) are elevated in chronic periodontitis patients compared with non-diseased subjects. The aim of the present study was to determine whether periodontal treatment could influence the level of serum antibodies to human HSP60 and Porphyromonas gingivalis GroEL, a bacterial homologue of human HSP60. Sera were obtained from 21 patients with moderate to advanced chronic periodontitis at the baseline examination and again after completion of treatment. Antibody levels were determined using an enzyme-linked immunosorbent assay. The mean anti-P. gingivalis GroEL antibody levels were down-regulated significantly by periodontal treatment when recombinant P. gingivalis GroEL was used as an antigen, whereas antibody levels to P. gingivalis GroEL-specific peptide were significantly elevated following successful periodontal therapy. The mean level of anti-human HSP60 antibody remained unchanged although individual levels of antibody either increased or decreased after periodontal treatment, suggesting that synthesis of these antibodies might be regulated independently during the course of periodontal infection. Although their regulatory mechanisms in chronic infection are not understood, further study would provide insight not only into the role of these antibodies in the pathogenesis of periodontitis but also into the possible link between periodontitis and systemic diseases such as coronary heart disease.

Clinical features and management of tumors affecting the lacrimal drainage apparatus

Purpose: To report the clinical features of a series of patients with lacrimal drainage apparatus tumors and present guidelines for management based on histopathology. Methods: A noncomparative retrospective chart review of the clinical, imaging, and pathologic findings of 37 patients presenting to four regional orbital Surgery departments with tumors affecting the lacrimal drainage apparatus between 1990 and 2004. Results: There were 37 patients, of whom 62% were male. The mean age at referral was 54 years. Epiphora, a palpable mass, and dacryocystitis were the most common presentations. Two thirds of the tumors were epithelial. with carcinomas being the most frequent (38%). followed by papillomas (27%). Lymphomas were the most common nonepithelial malignancy (30%). Epithelial tumors were more common in men (87%), whereas lymphomas were more common in women (57%). Treatment modalities included surgery, in addition to radiotherapy and/or chemotherapy and immunotherapy. Mean follow-up was 38 months. Thirty-three patients (89%) remain alive without evidence of disease and 4 patients died of recurrence and/or metastases. Conclusions: Lacrimal drainage apparatus tumors require careful initial management to ensure adequate local and systemic disease control. Atypical mucosa encountered during dacryocystorhinostomy should be biopsied and small papillomas or pedunculated tumors excised and analyzed with frozen sections. If a diffuse or infiltrative mass is encountered, it should be biopsied and managed on the basis of histopathology and extent of disease. Lymphomas should be treated according to protocols. whereas noninvasive carcinoma and extensive papillomas require complete excision of the system. Invasive disease requires en bloc excision. Long-term follow-up is essential for early detection of recurrence.

A study of potential serum markers for a diagnosis of gram-positive and gram-negative bacterial sepsis

Sepsis continues to be a major cause of morbidity and mortality as it can readily lead tosevere sepsis, septic shock, multiple organ failure and death. The onset can be rapid and difficult to define clinically. Despite the numerous candidate markers proposed in the literature, to date a serum marker for sepsis has not been found. The aim of this study was to assay the serum of clinically diagnosed patients with eithera Gram-negative or Gram- positive bacterial sepsis for elevated levels of nine potentialmarkers of sepsis, using commercially produced enzyme linked immunosorbent assays(ELISA). The purpose was to find a test marker for sepsis that would be helpful toclinicians in cases of uncertain sepsis and consequently expose false positive BC'scaused by skin or environmental contaminants. Nine test markers were assayed including IL-6, IL-I 0, ILI2, TNF-α, lipopolysaccharide binding protein, procalcitonin, sE-selectin, sICAM -1 and a potential differential marker for Gram-positive sepsis- anti-lipid S antibody. A total of 445 patients were enrolled into this study from the Queen Elizabeth Hospital and Selly Oak Hospital (Birmingham). The results showed that all the markers were elevated in patients with sepsis and that patients with a Gram-negative sepsis consistently produced higher median/range serum levels than those with a Gram-positive sepsis. No single marker was able to identify all the septic patients. Combining two markers caused the sensitivities and specificities for a diagnosis of sepsis to increase to within a 90% to 100% range. By a process of elimination the markers that survived into the last phase were IL-6 with sICAM -1, and anti-lipid S IgG assays Defining cut-off levels for a diagnosis of sepsis became problematic and a semi-blind trial was devised to test the markers in the absence of both clinical details and positive blood cultures. Patients with pyrexia of unknown origin and negative BC were included in this phase (4). The results showed that IL-6 with sICAM-l are authentic markers of sepsis. There was 82% agreement between the test marker diagnosis and the clinical diagnosis for sepsis in patients with a Gram-positive BC and 78% agreement in cases of Gram-negative Be. In the PUO group the test markers identified 12 cases of sepsis and the clinical diagnosis 15. The markers were shown to differentiate between early sepsis and sepsis, inflammatory responses and infection. Anti-lipid S with IL-6 proved be a sensitive marker for Gram-positive infections/sepsis.

The role of metabolism in the toxicity of N-alkylformamides

Using ionspray tandem mass spectrometry the glutathione conjugate SMG was identified as a biliary metabolite of DMF in rats (0.003% of a dose of 5OOmg/kg DMF i.p.). Formation of this metabolite was increased five fold after induction of CYP2E1 by acetone, and was inhibited to 20% of control values following pretreatment with disulfrram. Generation of SMG from DMF in vivo was shown to exhibit a large kinetic deuterium isotope effect (KWKD=10.1 ± 1.3), which most likely represents the product of 2 discrete isotope effects on N-demethylation and formyl oxidation reactions.The industrial solvent N,N-dimethylformamide (DMF) and the investigational anti-tumour agent N-methylformamide (NMF) cause liver damage in rodents and humans. The hepatotoxicity of N-alkylformamides is linked to their metabolism to N-alkylcarbamic acid thioesters. The enzymatic details of this pathway were investigated. Hepatocytes isolated from BALB/c mice which had been pretreated with acetone, an inducer of the cytochrome P-450 isozyme CYP2E1, were incubated with NMF (10mM). NMF caused extensive toxicity (> 90% ) as determined by lactate dehydrogenase (LDH) release, compared to cells from untreated animals. Incubation of liver cells with NMF for 6 hrs caused 60±17% LDH release whilst in the presence of DMSO (10mM), an alternative substrate for CYP2E1, LDH release was reduced to 20±10% . The metabolism of NMF to S-(N-methylcarbamoyl)glutathione (SMG) was measured in incubates with liver microsomes from mice, rats or humans. Metabolism of NMF was elevated in microsomes isolated from rats and mice pretreated with acetone, by 339% and 183% respectively. Pretreatment of animals with 4-methylpyrazole induced the metabolism of NMF to 280% by rat microsomes, but was without effect on NMF metabolism by mouse microsomes. The CYP2E1 inhibitors or alternative substrates diethyl dithiocarbamate (DEDTC), p-nitrophenol (PNP) and dimethyl sulphoxide (DMSO) strongly inhibited the metabolism of NMF in suspensions of rat liver microsomes, at concentrations which did not effect aminopyrine N-demethylation. The rate of metabolism of NMF to SMG in human microsomes correlated (r> 0.8) with the rate of metabolism of chlorzoxazone, a CYP2E1 probe. A polyclonal antibody against rat CYP2E1 (10mg/nmol P-450) inhibited NMF metabolism in microsomes from rats and humans by 75% and 80% , respectively. The amount of immunoblottable enzyme in human microsomes, determined using an anti-rat CYP2E1 antibody, correlated with the rate of NMF metabolism (r> 0.8). Purified rat CYP2E1 catalysed the generation of SMG from NMF. Formation of the DMF metabolite N-hydroxymethyl-N-methylformamide (HMMF) in incubations with rat liver microsomes was elevated by 200% following pretreatment of animals with acetone. Co-incubation with DEDTC (100μM) inhibited HMMF generation from DMF by 88% . Co-incubation of DMF (10mM) with NMF (1mM) inhibited the formation of SMG by 95% . A polyclonal antibody against rat CYP2E1 (10mg/nmol P-450) inhibited generation of HMMF in incubates with rat and human liver microsomes by 68.4% and 67.5% , respectively. Purified rat CYP2E1 catalysed the generation of HMMF from DMF. Using ionspray tandem mass spectrometry the glutathione conjugate SMG was identified as a biliary metabolite of DMF in rats (0.003% of a dose of 5OOmg/kg DMF i.p.). Formation of this metabolite was increased five fold after induction of CYP2E1 by acetone, and was inhibited to 20% of control values following pretreatment with disulfrram. Generation of SMG from DMF in vivo was shown to exhibit a large kinetic deuterium isotope effect (KHKD=10.1 ± 1.3), which most likely represents the product of 2 discrete isotope effects on N-demethylation and formyl oxidation reactions.

Role of Ca2+ in proteolysis-inducing factor (PIF)-induced atrophy of skeletal muscle

Proteolysis-inducing factor (PIF) induces muscle loss in cancer cachexia through a high affinity membrane bound receptor. This study investigates the mechanism by which the PIF receptor communicates to intracellular signalling pathways. C2C12 murine myoblasts were used as a model using PIF purified from MAC16 tumours. Calcium imaging was determined using fura-4-acetoxymethyl ester (Fura-4-AM). PIF induced a rapid rise in Ca2 +i, which was completely attenuated by a anti-receptor antibody, or peptides representing 20 mers of the N-terminus of the PIF receptor. Other agents catabolic for skeletal muscle including angiotensin II (AngII) tumour necrosis factor-a (TNF-a) and lipopolysaccharide (LPS) also induced a rise in Ca2 +i, but this was not attenuated by anti-PIF-receptor antibody. The rise in Ca2 +i induced by PIF and AngII was completely attenuated by the Zn2 + chelator D-myo-inositol-1,2,6-triphosphate, and this was reversed by administration of exogenous Zn2 +. The Ca2 +i rise induced by PIF was independent of the presence of extracellular Ca2 +, and attenuated by the Ca2 + pump inhibitor thapsigargin, suggesting that the Ca2 +i rise was due to release from intracellular stores. This rise in Ca2 +i induced by PIF was attenuated by both the phospholipase C inhibitor U73122 and 2-APB, an inhibitor of the inositol 1,4,5-triphosphate receptor, suggesting the involvement of a G-protein. Binding of the PIF to its receptor in skeletal muscle triggers a rise in Ca2 +i, which initiates a signalling cascade leading to a depression in protein synthesis, and an increase in protein degradation.

Label-free immunoassay for porcine circovirus type 2 based on excessively tilted fiber grating modified with staphylococcal protein A

Using excessively tilted fiber grating (Ex-TFG) inscribed in standard single mode fiber, we developed a novel label-free immunoassay for specific detection of porcine circovirus type 2 (PCV2), which is a minim animal virus. Staphylococcal protein A (SPA) was used to modify the silanized fiber surface thus forming a SPA layer, which would greatly enhance the proportion of anti-PCV2 monoclonal antibody (MAb) bioactivity, thus improving the effectiveness of specific adsorption and binding events between anti-PCV2 MAbs and PCV2 antigens. Immunoassay experiments were carried out by monitoring the resonance wavelength shift of the proposed sensor under different PCV2 titer levels. Anti-PCV2 MAbs were thoroughly dissociated from the SPA layer by treatment with urea, and recombined to the SPA layer on the sensor surface for repeated immunoassay of PCV2. The specificity of the immunosensor was inspected by detecting porcine reproductive and respiratory syndrome virus (PRRSV) first, and PCV2 subsequently. The results showed a limit of detection (LOD) for the PCV2 immunosensor of ~9.371TCID50/mL, for a saturation value of ~4.801×103TCID50/mL, with good repeatability and excellent specificity.

Immunotherapy for the treatment of poor prognosis cancers

Cancer is amongst the leading causes of death worldwide and the number one cause in the developed world. Every year there are close to 10 million cancer related deaths and this corresponds to hundreds of millions of euro in health care costs and lost productivity, placing a substantial drain on the economy. The efficacy of traditional treatment modalities for cancer therapy, such as surgery, radiotherapy and chemotherapy has plateaued, and while they are undoubtedly effective at prolonging patient lifespan, there is a high rate of adverse side effects and fatal reoccurrence. Currently, there is a huge amount of interest in the areas of cancer immunosurveillance and cancer immuno-editing, which explain some of the complex interactions between the host immune system and cancer. If left unchecked, cancerous malignancies have the ability to generate an immunosuppressive microenvironment, effectively shielding themselves from elimination and promoting tumour growth and progression. To overcome this, the potential of the immune system must be harnessed and the work undertaken in this thesis sought to contribute to this goal. Focus was placed on using novel therapies, combining tumour ablation with immune-modulating antibodies to maximise tumour elimination in an immune dependent manner, to overcome immunosuppression and promote immune activation. Chapter 2 focuses on the use of ECT as a method of tumour ablation and its effects on the immune system. ECT proved to be effective at inhibiting the tumour growth both in vitro and in vivo, and conferred significant survival advantages in both small and large animal models. More importantly, ECT proved to cause tumour death in an immune dependent manner, displaying the hallmarks of Immunogenic Cell Death, increases in immune cell infiltration and generating tumour-specific immune responses. Chapter 3 focuses on combining ECT with immune checkpoint blockade inhibitors; anti- CTLA-4 and anti-PD-1. Both combinations proved to be effective at inhibiting both primary and distal tumour growth, indicating the generation of tumour specific immune responses and prolonged animal survival. In addition, the treatments caused increases in the levels of certain intra-tumoural immune cell subsets and modulated the cytokine profile of treated animals in a way that was favourable overall. Chapter 4 focuses on the combining ECT with an anti-iCOS agonist antibody, capable of causing immune co-stimulation. This novel combinational therapy proved to be the most effective by far, with a high cure rate achieved across a number of different in vivo tumour models. Total regression was seen in both primary and distal tumours, as well as spontaneous metastases, with the tumour specific immune response generated conferring total protection to animals on tumour rechallenge. Overall the data presented here adds further insight into the area of cancer immunotherapy with some of the novel combinational therapies demonstrating substantial clinic potential.

Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library.

B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of potential therapeutic interest. To generate anti-B7-H4 targeting reagents, we isolated antibodies by differential cell screening of a yeast-display library of recombinant antibodies (scFvs) derived from ovarian cancer patients and we screened for functional scFvs capable to interfere with B7-H4-mediated inhibition of antitumor responses. We found one antibody binding to B7-H4 that could restore antitumor T cell responses. This chapter gives an overview of the methods we developed to isolate a functional anti-B7-H4 antibody fragment.

Portable “lab-on-chip” platform for bovine mastitis diagnosis in raw milk

Tese de Doutoramento em Ciências Veterinárias, Especialidade de Ciências Biológicas e Biomédicas

Regulation of prostate cancer cell function by activators of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is a key regulator of cell energy homeostasis. More recently, it has become apparent that AMPK regulates cell proliferation, migration and inflammation. Previous evidence has suggested that AMPK may influence proliferation and invasion by regulating the pro-proliferative mitogen-activated protein kinases (MAPKs). However, the mechanisms underlying this crosstalk between AMPK and MAPK signalling are not fully understood. As AMPK activation has been reported to have anti-proliferative effects, there has been increasing interest in AMPK activation as a therapeutic target for tumourigenesis. The aim of this study was to investigate whether AMPK activation influenced prostate cancer (PC) cell line proliferation, migration and signalling. Therefore, different PC cell lines were incubated with two structurally-unrelated molecules that activate AMPK by different mechanisms, AICAR and A769662. Both chemicals activated AMPK in a concentration- and time-dependent manner in PC3, DU145 and LNCaP cell lines. AMPK activity as assessed by AMPK activating phosphorylation as well as phosphorylation of the AMPK substrate ACC increased along with tumour severity in PC biopsies. Furthermore, both activators of AMPK decreased cell proliferation and migration in the androgen-independent PC cell lines PC3 and DU145. Inhibition of proliferation by A769662 was attenuated in AMPK α1-/- AMPK α2-/- knockout (KO) mouse embryonic fibroblasts (MEFs) compared to wild type (WT) MEFs, and the inhibitory effect on migration of AICAR lost significance in PC3 cells infected with adenoviruses expressing a dominant negative AMPK α mutant, indicating these effects are partially mediated by AMPK. Furthermore, long-term activation of AMPK was associated with inhibition of both the phosphatidylinositol 3’-kinase/protein kinase B (PI3K/Akt) signalling pathway in addition to the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathway. Indeed, the actions of AMPK activators on PC cell line viability were mimicked by selective inhibitors of Akt and ERK1/2 pathways. In contrast to the effects of prolonged incubation with AMPK activators, short-term incubation with AMPK activators had no effect on epidermal growth factor (EGF)-stimulated ERK1/2 phosphorylation in PC cell lines. In addition, AMPK activation did not influence phosphorylation of the other MAPK family members p38 and JNK. Interestingly, both AICAR and A769662 decreased EGF-stimulated ERK5 phosphorylation in PC3, DU145 and LNCaP cells as assessed with an anti-phospho-ERK5 antibody. Further characterisation of this effect indicated that prior stimulation with the AMPK activators had no effect on ERK5 phosphorylation stimulated by transient transfection with a constitutively active ERK5 kinase (MEK5DD), which represents the only known canonical kinase for ERK5. Intriguingly, the pattern of EGF-stimulated ERK5 phosphorylation was distinct from that mediated by MEK5DD activation of ERK5. This finding indicates that AMPK activation inhibits EGF-stimulated ERK5 phosphorylation at a point at or above the level of MEK5, although why EGF and constitutively active MEK5 stimulate markedly different immunoreactive species recognised by the anti-phospho-ERK5 antibody requires further study. A769662 had a tendency to reduce EGF-stimulated ERK5 phosphorylation in WT MEFs, yet was without effect in MEFs lacking AMPK. These data indicate that AMPK may underlie the effect of A769662 to reduce EGF-stimulated ERK5 phosphorylation. Prolonged stimulation of PC cell lines with AICAR or A769662 inhibited EGF-stimulated Akt Ser473 phosphorylation, whereas only incubation with A769662 rapidly inhibited Akt phosphorylation. This difference in the actions of the different AMPK activators may suggest an AMPK-independent effect of A769662. Furthermore, AICAR increased phosphorylation of Akt in WT MEFs, an effect that was absent in MEFs lacking AMPK, indicating that this effect of AICAR may be AMPK-dependent. Taken together, the data presented in this study suggest that AMPK activators markedly inhibit proliferation and migration of PC cell lines, reduce EGF-stimulated ERK1/2 and Akt phosphorylation after prolonged incubation and rapidly inhibit ERK5 phosphorylation. Both AMPK activators exhibit a number of effects that are likely to be independent of AMPK in PC cell lines, although inhibition of ERK1/2, ERK5 and Akt may underlie the effects of AMPK activators on proliferation, viability and migration. Further studies are required to understand the crosstalk between those signalling pathways and their underlying significance in PC progression.

HtrA, RseP, and Tsp do not elicit a pathology related serum IgG response during sexually transmitted infection with Chlamydia trachomatis

Chlamydia trachomatis sexually transmitted infection can cause serious reproductive morbidities. This study determined the prevalence of serum IgG response to C. trachomatis putative stress response proteins in females to test for an association with genital tract pathology. There was no significant association of serum IgG to HtrA, Tsp, or RseP with infection or pathology. cHSP60 serum IgG prevalence was significantly associated with infection compared to negative (infertile) controls (p = 0.002), but not with upper genital tract pathology. Serum IgG1-4 antibody subclasses reactive with the antigens was not significantly different between cohorts, although different responses to each antigen were detected.

CDKN2A is not the principal target of deletions on the short arm of chromosome 9 in neuroendocrine (Merkel cell) carcinoma of the skin

The majority of small-cell lung cancers (SCLCs) express p16 but not pRb. Given our previous study showing loss of pRb in Merkel cell carcinoma (MCC)/neuroendocrine carcinoma of the skin and the clinicopathological similarities between SCLC and MCC, we wished to determine if this was also the case in MCC. Twenty-nine MCC specimens from 23 patients were examined for deletions at 10 loci on 9p and 1 on 9q. No loss of heterozygosity (LOH) was seen in 9 patients including 2 for which tumour and cell line DNAs were examined. Four patients had LOH for all informative loci on 9p. Ten tumours showed more limited regions of loss on 9p, and from these 2 common regions of deletion were determined. Half of all informative cases had LOH at D9S168, the most telomeric marker examined, and 3 specimens showed loss of only D9S168. A second region (IFNA-D9S126) showed LOH in 10 (44%) cases, and case MCC26 showed LOH for only D9S126, implicating genes centromeric of the CDKN2A locus. No mutations in the coding regions of p16 were seen in 7 cell lines tested, and reactivity to anti-p16 antibody was seen in all 11 tumour specimens examined and in 6 of 7 cell lines from 6 patients. Furthermore, all cell lines examined reacted with anti-p14(ARF) antibody. These results suggest that neither transcript of the CDKN2A locus is the target of deletions on 9p in MCC and imply the existence of tumour-suppressor genes mapping both centromeric and telomeric of this locus.

Angiogenesis is an independent prognostic factor in malignant mesothelioma

Angiogenesis is essential for tumour growth beyond 1 to 2 mm in diameter. The clinical relevance of angiogenesis, as assessed by microvessel density (MVD), is unclear in malignant mesothelioma (MM). Immunohistochemistry was performed on 104 archival, paraffin-embedded, surgically resected MM samples with an anti-CD34 monoclonal antibody, using the Streptavidin-biotin complex immunoperoxidase technique. 93 cases were suitable for microvessel quantification. MVD was obtained from 3 intratumoural hotspots, using a Chalkley eyepiece graticule at × 250 power. MVD was correlated with survival by Kaplan-Meier and log-rank analysis. A stepwise, multivariate Cox model was used to compare MVD with known prognostic factors and the EORTC and CALGB prognostic scoring systems. Overall median survival from the date of diagnosis was 5.0 months. Increasing MVD was a poor prognostic factor in univariate analysis (P = 0.02). Independent indicators of poor prognosis in multivariate analysis were non-epithelial cell type (P = 0.002), performance status > 0 (P = 0.003) and increasing MVD (P = 0.01). In multivariate Cox analysis, MVD contributed independently to the EORTC (P = 0.006), but not to the CALGB (P = 0.1), prognostic groups. Angiogenesis, as assessed by MVD, is a poor prognostic factor in MM, independent of other clinicopathological variables and the EORTC prognostic scoring system. Further work is required to assess the prognostic importance of angiogenic regulatory factors in this disease. © 2001 Cancer Research Campaign.

Expression of β1 integrin in laminin-adhesion-selected human colon cancer cell lines of varying tumorigenicity

Laminin has been shown to promote the malignant phenotype and the expression of certain laminin receptors has been correlated with the malignant character of the tumors. Here new cell lines were isolated from a human colon cancer cell line (LCC-C1) based on their adhesiveness to laminin. The laminin-adherent subclone formed large tumors in nude mice, whereas the laminin-nonadherent subclone failed to form sizable tumors. Only the laminin-adherent subclone adhered to laminin and invaded through Matrigel-coated filters. The adhesive and invasive ability of the cells was almost completely blocked by low concentrations (1.0 μg/ml) of anti-β1 integrin antibody. The amounts of total cellular β1 integrin protein were similar in the two subclones when compared by Western blot, and the mRNA levels also did not differ. The localization of β1 integrin laminin receptor varied in the two subclones; the laminin-adherent subclone showed a linear distribution along the cell-cell junctions, while the laminin-nonadherent subclone did not stain between the cells. Using laminin-Sepharose affinity chromatography, more β1 integrin was obtained from the laminin-adherent subclone. These findings suggest that alterations in the affinity of β1 integrin for laminin and in its membrane distribution might be involved in the increased tumorigenicity observed in colon cancer cells.