T and B Cell Composition and Cytokine Producing Capacity Before and After Bariatric Surgery.

Morbid obesity is associated with a chronic state of low-grade inflammation, which may lead to accelerated differentiation of T and B cells. These differentiated immune cells are strongly cytotoxic and have an increased pro-inflammatory cytokine producing capacity. Furthermore, the anti-inflammatory function of the T and B cells decreases. The aim of this study was to evaluate the effect of morbid obesity on the subset profile and cytokine producing capacity of T and B cells. Subsequently, we assessed whether bariatric surgery affected the subset profile and cytokine producing capacity of these cells. We determined the proportion of T and B cell subsets and their cytokine producing capacity in peripheral blood collected from 23 morbidly obese patients before and three months after bariatric surgery using flow-cytometry. We compared this with the results of 25 lean controls. Both CD4+ and CD8+ T cells showed a more differentiated subset profile in morbidly obese patients as compared to lean controls, which was not recovered three months after bariatric surgery. The B cell composition of morbidly obese patients after bariatric surgery adjusted towards the profile of lean controls. However, the IL-2 and IFN-γ producing capacity of CD8+ T cells and the IL-2, IFN-γ, TNF-α and IL-10 producing capacity of B cells was not restored three months after bariatric surgery. In conclusion, the data suggest that the immune system has the capacity to recover from the detrimental effects of morbid obesity within three months after bariatric surgery in terms of cell composition; however, this was not seen in terms of cytokine producing capacity. The full restoration of the immune system after bariatric surgery may thus take longer.

Inhibition of activin-like kinase 4/5 attenuates cancer cachexia associated muscle wasting.

Cancer mediated activation of the ActRIIB-ALK4/5 heterodimer by myostatin is strongly associated with muscle wasting. We investigated in vitro and in vivo the efficacy of ALK4/5 receptor blockers SB431542 and GW788388 in preventing muscle wasting, and explored synergy with IGF-I analogue LONG R3 (LR3) IGF-I. In vitro, C2C12 skeletal muscle cells were treated with vehicle, SB431542, GW788388 and LR3 IGF-I. A C26-CD2F1 cachexia model was used to induce cachexia in vivo. Mice were allocated as non-tumour bearing (NTB) or C26 tumour-bearing (C26 TB) vehicle control, treated with SB431542, LR3 IGF-I, SB431542 and LR3 IGF-I, or GW788388 (intraperitoneally or orally). In vitro, differentiation index and mean nuclei count increased using SB431542, GW788388, LR3 IGF-I. In vivo, GW788388 was superior to SB431542 in limiting loss of bodyweight, grip-strength and gastrocnemius weight. and downregulated Atrogin-1 expression comparable to NTB mice. LR3 IGF-I treatment limited loss of muscle mass, but at the expense of accelerated tumour growth. In conclusion, treatment with GW788388 prevented cancer cachexia, and downregulated associated ubiquitin ligase Atrogin-1.

A signature of renal stress resistance induced by short-term dietary restriction, fasting, and protein restriction.

During kidney transplantation, ischemia-reperfusion injury (IRI) induces oxidative stress. Short-term preoperative 30% dietary restriction (DR) and 3-day fasting protect against renal IRI. We investigated the contribution of macronutrients to this protection on both phenotypical and transcriptional levels. Male C57BL/6 mice were fed control food ad libitum, underwent two weeks of 30%DR, 3-day fasting, or received a protein-, carbohydrate- or fat-free diet for various periods of time. After completion of each diet, renal gene expression was investigated using microarrays. After induction of renal IRI by clamping the renal pedicles, animals were monitored seven days postoperatively for signs of IRI. In addition to 3-day fasting and two weeks 30%DR, three days of a protein-free diet protected against renal IRI as well, whereas the other diets did not. Gene expression patterns significantly overlapped between all diets except the fat-free diet. Detailed meta-analysis showed involvement of nuclear receptor signaling via transcription factors, including FOXO3, HNF4A and HMGA1. In conclusion, three days of a protein-free diet is sufficient to induce protection against renal IRI similar to 3-day fasting and two weeks of 30%DR. The elucidated network of common protective pathways and transcription factors further improves our mechanistic insight into the increased stress resistance induced by short-term DR.

Gene therapy in in vivo isolated perfusion models.

Locoregional administration of a genetic construct by means of in vivo, in situ isolated perfusion (IP) of a target organ or extremity is a method that may increase in vivo efficacy. Vascular isolation and perfusion minimizes systemic exposure and thereby reduces unwanted side effects. Isolated hepatic perfusion (IHP) is the most extensively studied IP model, especially in gene therapy protocols for inborn errors of metabolism. To achieve stable transduction most frequently retroviruses are used in IHP. IHP is combined with hepatectomy or vascular ligation of liver lobes to induce liver regeneration increasing transduction efficacy. When adenoviruses are used in IHP high transduction percentages of hepatocytes can be achieved without significant toxicity. In tumor models adenoviral IHP has been performed, but has not been very successful up till now. Isolated limb perfusion (ILP) is a promising treatment modality in pre-clinical cancer gene therapy studies. After ILP a homogeneous distribution of transduced cells was demonstrated especially at the viable rim of the tumor and around tumor associated vessels. Moreover complete tumor responses have been observed. Isolated pulmonary perfusion (IPP) results in selective expression in the perfused lung and the duration of expression is longer than after systemic administration. In rats a significant decrease of tumor nodules upon IPP can be achieved. Furthermore other less studied perfusion models are discussed: isolated kidney perfusion (IKP), isolated spleen perfusion (ISP) and isolated cardiac perfusion (ICP). IP is a methodology that delivers vectors highly selectively, with a long exposure time and high concentrations at the target side. This results in higher transduction rates and thereby may improve therapeutic effects.

Impaired neutralising antibody formation and high transduction efficacy after isolated hepatic perfusion with adenoviral vectors.

Local adenoviral gene transfer can be performed by means of isolated hepatic perfusion (IHP). This methodology is a very effective and safe way to deliver adenoviral vectors. We studied the immune response after IHP. A decreased neutralising antibody formation was observed, offering possibilities for further research in the field of gene therapy in isolated perfusion settings.

Dynamic contrast-enhanced MRI using macromolecular contrast media for monitoring the response to isolated limb perfusion in experimental soft-tissue sarcomas.

The objective of this study was to evaluate the potential of dynamic contrast-enhanced MRI for quantitative characterization of tumor microvessels and to assess the microvascular changes in response to isolated limb perfusion with TNF-alpha and melphalan. Dynamic contrast-enhanced MRI was performed in an experimental cancer model, using a macromolecular contrast medium, albumin-(Gd-DTPA)45. Small fragments of BN 175, a soft-tissue sarcoma, were implanted in 11 brown Norway (BN) rats. Animals were assigned randomly to a control (Haemaccel) or drug-treated group (TNF-alpha/melphalan). MRI was performed at baseline and 24 h after ILP. The transendothelial permeability (K(PS)) and the fractional plasma volume (fPV) were estimated from the kinetic analysis of MR data using a two-compartment bi-directional model. K(PS) and fPV decreased significantly in the drug-treated group compared to baseline (p<0.05). In addition, K(PS) post therapy was significantly lower (p<0.05) in the drug-treated group than in the control group. There was no significant difference in fPV between the drug-treated and the control group after therapy. Tumor microvascular changes in response to isolated limb perfusion can be determined after 24 h by dynamic contrast-enhanced MRI. The data obtained in this experimental model suggest possible applications in the clinical setting, using the appropriate MR contrast agents.

Lack of efficacy of Doxil in TNF-alpha-based isolated limb perfusion in sarcoma-bearing rats.

Here we show that Doxil has minimal antitumour activity in the isolated limb perfusion (ILP) setting and its activity was not enhanced by the addition of tumour necrosis factor (TNF). Doxil accumulation in tumour tissue was low and also not augmented by TNF. In contrast, activity of free conventional doxorubicin was enhanced by TNF. We conclude that application of Doxil in a TNF-based ILP is not a useful alternative to free conventional doxorubicin or melphalan.

Degree of tumour vascularity correlates with drug accumulation and tumour response upon TNF-alpha-based isolated hepatic perfusion.

Isolated hepatic perfusion (IHP) with melphalan with or without tumour necrosis factor alpha (TNF-alpha) is currently performed in clinical trials in patients with hepatic metastases. Previous studies led to the hypothesis that the use of TNF-alpha in isolated limb perfusion causes specific destruction of tumour endothelial cells and thereby induces an increased permeability of tumour vasculature. However, whether TNF-alpha contributes to the therapeutic efficacy in IHP still remains unclear. In an in vivo rat liver metastases model we studied three different tumours: colon carcinoma CC531, ROS-1 osteosarcoma and BN-175 soft-tissue sarcoma which exhibit different degrees of vascularisation. IHP was performed with melphalan with or without the addition of TNF-alpha. IHP with melphalan alone resulted, in all tumour types, in a decreased growth rate. However in the BN-175 tumour addition of TNF-alpha resulted in a strong synergistic effect. In the majority of the BN-175 tumour-bearing rats, a complete response was achieved. In vitro cytoxicity studies showed no sensitivity (CC531 and BN-175) or only minor sensitivity (ROS-1) to TNF-alpha, ruling out a direct interaction of TNF-alpha with tumour cells. The response rate in BN-175 tumour-bearing rats when TNF-alpha was coadministrated with melphalan was strongly correlated with drug accumulation in tumour tissue, as only in these rats a five-fold increased melphalan concentration was observed. Secondly, immunohistochemical analysis of microvascular density (MVD) of the tumour showed a significantly higher MVD for BN-175 tumour compared to CC531 and ROS-1. These results indicate a direct relation between vascularity of the tumour and TNF-alpha mediated effects. Assessment of the tumour vasculature of liver metastases would be a way of establishing an indication for the utility of TNF-alpha in this setting.

Prerequisites for effective adenovirus mediated gene therapy of colorectal liver metastases in the rat using an intracellular neutralizing antibody fragment to p21-Ras.

Ras mutations are present in 40-50% of colorectal cancers. Inactivating this oncogene may therefore reduce proliferation capacity. In order to target ras we studied the transduction efficacy and anti tumour activity of an adenoviral vector expressing an intracellular, neutralizing single chain antibody to p21-ras (Y28). In in vitro studies transfection levels of the K-ras mutated rat colon carcinoma cell line CC531 were studied using the LacZ marker gene. In our in vivo liver metastases model different routes of administration were evaluated to determine which regimen resulted in the best transfection levels and tumour responses: intravenous injection, intratumoural injection, isolated liver perfusion, or hepatic artery infusion. CC531 cells are readily transfected in vitro, resulting in significant inhibition of tumour cell proliferation by the Y28 construct. Intravenous injection did not result in any measurable transfection. Intratumoural injection resulted only in the transfection of tumour cells along the needle track. IHP as well as single HAI achieved low transfection levels of tumour tissue. Expression of Y28 was demonstrated in tumours after IT injection, HAI and IHP. Whereas, repeated HAI's clearly achieved expression in and around tumour associated vessels. Only five times repeated HAI's with Y28 resulted in a tumour response: in all animals tumour growth was inhibited, and in three rats out of eight a complete regression of the liver tumours was observed.