Martin B. Oleksiewicz

Anti-bacterial monoclonal antibodies: Back to the future?

Todays medicine has to deal with the emergence of multi-drug resistant bacteria, and is beginning to be confronted with pan-resistant microbes. This worsening inadequacy of the antibiotics concept, which has ruled infectious medicine in the last six decades creates an increasing unmet medical need that can be addressed by passive immunization. While past experience from the pre-antibiotic era with serum therapy was in many cases encouraging, antibacterial monoclonal antibodies have so far suffered high attrition rates in the clinic, generally from lack of efficacy. Yet, we believe that recent developments in a number of areas such as infectious disease pathogenesis research, translational medicine, mAb engineering, mAb manufacturing and rapid bedside diagnostics are converging to make the medium-term future permissive for antibacterial mAb development. Here, we review antibacterial mAb-based approaches that are or were in clinical development, and may potentially act as paradigms with regards to molecular targets, antibody formats and mode-of-action, pre-clinical validation and selection of most relevant patient populations, in order to increase the likelihood of successful product development in this field.

Rat Urinary Bladder Carcinogenesis by Dual-Acting PPARalpha + gamma Agonists.

Despite clinical promise, dual-acting activators of PPARα and γ (here termed PPARα+γ agonists) have experienced high attrition rates in preclinical and early clinical development, due to toxicity. In some cases, discontinuation was due to carcinogenic effect in the rat urothelium, the epithelial layer lining the urinary bladder, ureters, and kidney pelvis. Chronic pharmacological activation of PPARα is invariably associated with cancer in rats and mice. Chronic pharmacological activation of PPARγ can in some cases also cause cancer in rats and mice. Urothelial cells coexpress PPARα as well as PPARγ, making it plausible that the urothelial carcinogenicity of PPARα+γ agonists may be caused by receptor-mediated effects (exaggerated pharmacology). Based on previously published mode of action data for the PPARα+γ agonist ragaglitazar, and the available literature about the role of PPARα and γ in rodent carcinogenesis, we propose a mode of action hypothesis for the carcinogenic effect of PPARα+γ agonists in the rat urothelium, which combines receptor-mediated and off-target cytotoxic effects. The proposed mode of action hypothesis is being explored in our laboratories, towards understanding the human relevance of the rat cancer findings, and developing rapid in vitro or short-term in vivo screening approaches to faciliate development of new dual-acting PPAR agonist compounds.

Agonism and Antagonism at the Insulin Receptor

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (3H-thymidine incorporation), and not on metabolic endpoints (14C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (3H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.

High frequency of tumor cells with nuclear Egr-1 protein expression in human bladder cancer is associated with disease progression

BackgroundEgr-1 (early growth response-1 transcription factor) has been proposed to be involved in invasion and metastasis processes of human bladder cancer, but Egr-1 protein expression levels in human bladder cancer have not been investigated. In the present study we investigated the expression levels of Egr-1 protein in early stages of human bladder cancer and correlated it to later progression.MethodsExpression of Egr-1 protein in human bladder cancer was examined by immunohistochemistry, on a tissue microarray constructed from tumors from 289 patients with non-muscle invasive urothelial bladder cancer.ResultsThe frequency of tumor cells with nuclear Egr-1 immunolabelling correlated to bladder cancer stage, grade and to later progression to muscle-invasive bladder cancer (T2-4). Stage T1 tumors exhibited significantly higher frequencies of tumor cells with nuclear Egr-1 immunolabelling than Ta tumors (P = 0.001). Furthermore, Kaplan-Meier survival analysis showed that a high frequency of tumor cells with nuclear Egr-1 immunolabelling was significantly associated with a higher risk of progression to stage T2-4 (log-rank test, P = 0.035). Tumor cells with nuclear Egr-1 immunolabelling were found to localize at the tumor front in some of the tumor biopsies.ConclusionThe results from this study support a potential involvement of Egr-1 in the progression from non-muscle invasive bladder cancers to muscle invasive bladder cancer.

Synchronization in G0/G1 enhances the mitogenic response of cells overexpressing the human insulin receptor A isoform to insulin

Evaluating mitogenic signaling specifically through the human insulin receptor (IR) is relevant for the preclinical safety assessment of developmental insulin analogs. It is known that overexpression of IR sensitizes cells to the mitogenic effects of insulin, but it is essentially unknown how mitogenic responses can be optimized to allow practical use of such recombinant cell lines for preclinical safety testing. We constitutively overexpressed the short isoform of the human insulin receptor (hIR-A, exon 11-negative) in L6 rat skeletal myoblasts. Because the mitogenic effect of growth factors such as insulin is expected to act in G0/G1, promoting S-phase entry, we developed a combined topoinhibition + serum deprivation strategy to explore the effect of G0/G1 synchronization as an independent parameter in the context of serum deprivation, the latter being routinely used to reduce background in mitogenicity assays. G0/G1 synchronization significantly improved the mitogenic responses of L6-hIR cells to insulin, measured by 3H-thymidine incorporation. Comparison with the parental L6 cells using phospho-mitogen-activated protein kinase, phospho-AKT, as well as 3H-thymidine incorporation end points supported that the majority of the mitogenic effect of insulin in L6-hIR cells was mediated by the overexpressed hIR-A. Using the optimized L6-hIR assay, we found that the X-10 insulin analog was more mitogenic than native human insulin, supporting that X-10 exhibits increased mitogenic signaling through the hIR-A. In summary, this study provides the first demonstration that serum deprivation may not be sufficient, and G0/G1 synchronization may be required to obtain optimal responsiveness of hIR-overexpressing cell lines for preclinical safety testing.

Trans-Species Comparison of PPAR and RXR Expression by Rat and Human Urothelial Tissues

Because some investigational peroxisome proliferator-activated receptors (PPAR) agonists cause tumors in the lower urinary tract of rats, we compared normal human and rat urothelium in terms of PPAR and retinoid X receptor (RXR) expression and proliferation-associated phenotypes. In situ, few human but most rat urothelial cells were Ki67 positive, indicating fundamental differences in cell cycle control. Rat and human urothelia expressed all 3 PPAR and the RXRα and RXRβ isoforms in a predominantly nuclear localization, indicating that they may be biologically active. However, immunolocalization differences were observed between species. First, whereas PPARα and PPARβ/δ were expressed throughout the human bladder or ureteric urothelium, in the rat urothelium PPARα was primarily, and PPARβ/δ exclusively, restricted to superficial cells. Second, RXRβ was restricted to intermediate and superficial layers of the human urothelium but tended to be absent from the rat superficial cells. Third, PPARγ expression was present throughout the urothelia of both species but was most intense in the superficial human urothelium. Species differences were also observed in the expression of PPAR and RXR isoforms between cultured rat and human urothelial cells and in the smooth muscle. Our findings highlight the unique coexpression of multiple PPAR and RXR isoforms by urothelium and suggest that species differences in PPAR function between rat and human urothelia may be explored in an in vitro setting.

Identification of stable and oestrus cycle-independent housekeeping genes in the rat mammary gland and other tissues

The function and development of the rat mammary gland is dependent on the oestrus cycle. Normalization of gene expression in mammary gland samples assessed by quantitative RT-PCR therefore requires housekeeping genes (HKGs) which are stably expressed during the oestrus cycle. mRNA expression of 10 HKGs was measured in the rat mammary gland at different phases of the oestrus cycle. In addition, mRNA expression of the HKGs was measured in a panel of other rat tissues comprising laser microdissected mammary gland alveolar lobules and interlobular connective tissue and macrodissected mammary gland, liver, skeletal muscle, colon and ovary samples. Expression and ranking of HKGs varied between tissues and oestrus cycle phases and several HKGs were necessary for normalization between samples. In the mammary gland samples, three HKGs (Sdha, Tbp, and Atp5b) were identified as the optimal combination of stably expressed genes across oestrus cycle phases. For normalization between samples from the entire panel of rat tissues, eight HKGs (Rps18, Eef1a1, B2m, Actb, Tbp, Hprt, Pgk1, and Sdha) were identified as the optimal combination. These HKGs are of general relevance for studies comparing gene expression between different rat tissues.

An alternative method for preparation of tissue sections from the rat mammary gland.

In toxicopathological studies of the rat mammary glands, the guidelines of the Registry of Industrial Toxicology and Animal Data (RITA) recommend transverse sections of the inguinal mammary gland. However, occasionally limited amounts of mammary gland tissue are found in transverse sections. We compared transverse sectioning with an alternative method comprising horizontal sections of the rat mammary glands. Normal cycling female Sprague Dawley rats were sacrificed in proestrous, estrous, metestrous and diestrous, and samples from all mammary glands were collected. Transverse sections were prepared from the left-sided glands, and horizontal sections were cut from the right-sided glands. Sections were stained with HE, and epithelial and myoepithelial cells were visualized by immunohistochemical staining of cytokeratin 18 and alpha smooth muscle actin, respectively. Area of the mammary fat pad, mammary epithelium and connective tissue were measured in randomly sampled vision fields from each section. Horizontal sections contained a significantly larger area of mammary fat pad as well as glandular and connective tissue. No differences in tissue densities of epithelial or myoepithelial cells or connective tissue were observed between transverse sections and horizontal sections. Interestingly, densities of epithelium and connective tissue varied between cranial and caudal glands as well as the phases of the estrous cycle. In conclusion, horizontal histological sections of the rat mammary gland provided significantly larger samples of mammary gland tissue with no difference in tissue composition compared to transverse sections, which are recommended in the RITA guidelines.

Mammary gland proliferation in female rats: Effects of the estrous cycle, pseudo-pregnancy and age

Assessment of mammary gland proliferation in rats is an important endpoint in preclinical safety studies of pharmaceutical compounds. However, existing data on mammary gland proliferation in rats during the estrous cycle is conflicting, and it is unknown whether mammary gland proliferation differs between young and mature female virgin rats. Additionally, it is unclear which of the commonly applied markers of proliferating cells that is optimal for assessment of rat mammary gland proliferation. In this study the caudal thoracic, the abdominal and the cranial inguinal (i.e., the 3rd the 4th and the 5th) mammary gland were collected from 29 young and 26 mature non-treated, virgin female Sprague Dawley rats. Estrous cycle stage was determined from repeated vaginal smears and histological examination of the reproductive organs. Proliferation of mammary epithelium was assessed by immunohistochemistry using three markers: PCNA, Ki67, and BrdU. Proliferation of the mammary epithelium occurred mainly in the terminal end buds in the young animals. Epithelial proliferation was significantly increased during metestrus compared to the other phases. Mammary gland proliferation in pseudo-pregnant females was increased compared to proestrus, estrus and diestrus, but not metestrus. Except during estrus no difference in mammary gland proliferation was observed between young and mature female rats, and no significant differences was observed between different mammary glands. The percentages of PCNA-, Ki67- and BrdU-positive epithelial cells were significantly correlated. In conclusion, the variation in normal proliferation between estrous cycle stages and animals with an irregular estrous cycle should be considered in toxico-pathological studies of mammary gland proliferation.

Unique expression pattern of the three insulin receptor family members in the rat mammary gland: dominance of IGF-1R and IRR over the IR, and cyclical IGF-1R expression.

Supra‐pharmacological doses of the insulin analog X10 (AspB10) increased the incidence of mammary tumors in female Sprague–Dawley rats in chronic toxicity studies, most likely via receptor‐mediated mechanisms. However, little is known about the expression of the insulin receptor family in the rat mammary gland. Using laser micro‐dissection, quantitative RT‐PCR and immunohistochemistry, we examined the expression of IR (insulin receptor), IGF‐1R (IGF‐1 receptor), IRR (insulin receptor‐related receptor), ERα (estrogen receptor alpha), ERβ (estrogen receptor beta) and PR (progesteron receptor) in young, virgin, female Sprague–Dawley rats and compared to expression in reference organs. The mammary gland displayed the highest expression of IRR and IGF‐1R. In contrast, low expression of IR transcripts was observed in the mammary gland tissue with expression of the IR‐A isoform being 5‐fold higher than the expression of the IR‐B. By immunohistochemistry, expression of IR and IGF‐1R was detected in all mammary gland epithelial cells. Expression of ERα and PR was comparable between mammary gland and ovary, whereas expression of ERβ was lower in mammary gland than in the ovary. Finally, expression of IGF‐1R and PR in the mammary gland varied during the estrous cycle. These findings are important for the understanding of carcinogenic effects of insulin analogs in the rat mammary gland, and relevant for development of refined short‐term models for preclinical safety assessment of insulin analogs. Copyright