Bedside to bench and back again: how animal models are guiding the development of new immunotherapies for cancer

Immunotherapy using adoptive cell transfer is a promising approach that can result in the regression of bulky, invasive cancer in some patients. However, currently available therapies remain less successful than desired. To study the mechanisms of action and possible improvements in cell-transfer therapies, we use a murine model system with analogous components to the treatment of patients. T cell receptor transgenic CD8+ T cells (pmel-1) specifically recognizing the melanocyte differentiation antigen gp100 are adoptively transferred into lympho-depleted mice bearing large, established, 14-day subcutaneous B16 melanoma (0.5-1 cm in diameter) on the day of treatment. Adoptive cell transfer in combination with interleukin interleukin-2 or interleukin-15 cytokine administration and vaccination using an altered form of the target antigen, gp100, can result in the complete and durable regression of large tumor burdens. Complete responders frequently develop autoimmunity with vitiligo at the former tumor site that often spreads to involve the whole coat. These findings have important implications for the design of immunotherapy trials in humans.     

Peptide analysis,stability studies,and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-A(g7)

The MHC class II molecule H2-A(g7) is the chief genetic determinant in insulin-dependent diabetes mellitus of the non-obese diabetic (NOD) mice. Poor peptide binding ability, as well as presentation of a unique subset of peptides by this molecule was suggested to promote autoimmunity in this strain. However, several laboratories have presented results in favor of an H2-A(g7) molecule that can avidly bind many different peptides. The crystal structures of H2-A(g7) in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2-A(g7), we eluted natural ligands from purified H2-A(g7) molecules isolated from the H2-A(g7)-transfected M12-C3 cells. A low peptide yield dominated by a few peptide ligands was found. Pool sequencing and alignment of individual ligands on the basis of molecular modeling revealed a peptide-binding motif with basic/aliphatic/small hydrophilic amino acids at relative position 1 (p1), aliphatic amino acids at p4, Ala at p6, and acidic amino acids and Ser/Gly at p9, as well as acidic residues at p10/11. Though weak, the binding of individual ligands, as well as the importance of an acidic C-terminal residue was confirmed by peptide binding studies to isolated H2-A(g7) molecules. Furthermore, the H2-A(g7) molecule incompletely dissociated into its constituent chains in SDS-electrophoresis under nonreducing conditions. This provides additional evidence of its weak affinity for peptides, which probably arises from the combination of beta56His/beta57Ser/beta78Ala and other unique H2-A(g7) residues in contact with the antigenic peptide. These results allow a better understanding of the role of this molecule in the development of autoimmunity and the identification of epitopes relevant to diabetes.     

Impaired sodium excretion, decreased glomerular filtration rate and elevated blood pressure in endothelin receptor type B deficient rats

The renal endothelin (ET) system, particularly the ET type B receptor, has been implicated in the regulation of sodium excretion and glomerular filtration rate (GFR). We analyzed kidney morphology and function in a rat strain characterized by complete absence of a functional ETB receptor. Due to Hirschsprung's disease limiting lifetime in these rats, studies were performed in 23-day-old rats. Kidney size and morphology (glomerular and interstitial matrix content, glomerular size and cell density and intrarenal vascular morphology) were normal in ETB-deficient rats. There were also no evidence of altered kidney cell cycle regulation in these rats. GFR was significantly lower, by 72% (P<0.001), in homozygous ETB-deficient rats than in wild-type rats. Fractional sodium excretion was likewise markedly reduced by 84% in homozygous ETB-deficient rats (P<0.001 versus wild-type rats). Treatment with the specific epithelial sodium channel blocker amiloride led to a much higher increase in fractional sodium excretion in ETB-deficient rats (934.2+/-73% in ETB-deficient rats versus 297+/-20% in wild-type rats, expressed as percentage of corresponding placebo treated control; P<0.001). Mean arterial blood pressure was elevated by 7.9 mmHg in homozygous ETB-deficient rats (P<0.05 versus wild-type rats). Our study demonstrates that ETB-deficiency causes early onset kidney dysfunction characterized by a markedly reduced sodium excretion, decreased GFR, and slightly elevated blood pressure. The complete absence of the ETB receptor causes in the kidney--in contrast to the colon--a functional rather than a developmental, neural crest cell dependent disease, since kidney morphology was normal in ETB-deficient rats. The much higher increase in the fractional sodium excretion in ETB-deficient rats after pharmacological blockade of the epithelial sodium channel indicates that the decreased fractional sodium excretion in ETB-deficient rats is most probably due to a lack of the inhibitory property of the ETB receptor on the epithelial sodium channel activity.     

Effects of combined renovascular hypertension and diabetes mellitus on myocardial cells,non-vascular interstitium and capillaries: a stereological study on rat hearts

Summary The effects of combined renovascular hypertension and diabetes mellitus on the rat heart were investigated in order to detect possible synergistic effects of the two conditions. Hypertensive diabetic and hypertensive non-diabetic animals were compared to diabetic and non-diabetic controls. Hypertension was established for 12 weeks by a surgical stenosis of the left renal artery; diabetes mellitus was maintained for 8 weeks by a single intraperitoneal injection of 60 mg/kg streptozotocin. Light microscopic stereology did not reveal significant divergences between diabetic hypertensives and non-diabetic hypertensives. Hypertension induced a focal perivascular and interstitial fibrosis with increased volume densities of non-vascular interstitium and fibrosis (P<0.001). Capillary density (Q_A) was decreased in transverse sections (P<0.01) and increased in longitudinal sections (P<0.01). This indicates a three-dimensional remodelling of the capillary bed with an increased number of obliquely running capillaries. At least the length density (L_V) of capillaries (mm/mm³) tends to be normalized in long-term renovascular hypertension. At the ultrastructural level, a synergism of hypertension and diabetes mellitus was observed: the volume ratio of mitochondria to myofibrils was significantly decreased in hypertensive diabetics, but not in non-diabetic hypertensives or in diabetics. This may enhance the risk of cardiac deterioration. We conclude that the primary target of the synergistic damage in hypertensive diabetic heart muscle disease is the myocardial cell and not the cardiac interstitium.Preliminary results of this study have been published in: Mall G (1991) Morphometric study on the rat heart in combined renovascular hypertension and diabetes mellitus. In: Nagano N, Dhalla NS (eds) The diabetic heart. Raven Press, New York, pp 115–124Dedicated to Prof. Dr. med. G. Seifert on the occasion of his 70th birthday     

Amygdala-prefrontal coupling depends on a genetic variation of the serotonin transporter

Major depression is conditionally linked to a polymorphism of the human serotonin transporter gene (SLC6A4). During the presentation of aversive, but not pleasant, pictures, healthy carriers of the SLC6A4 short (s) allele showed stronger activation of the amygdala on functional magnetic resonance imaging. s carriers also showed greater coupling between the amygdala and the ventromedial prefrontal cortex, which may contribute to the abnormally high activity in the amygdala and medial prefrontal cortex seen in major depression.     

Human defensins

Antimicrobial peptides are small, cationic, amphiphilic peptides of 12–50 amino acids with microbicidal activity against both bacteria and fungi. The eukaryotic antimicrobial peptides may be divided into four distinct groups according to their structural features: cysteine-free -helices, extended cysteine-free -helices with a predominance of one or two amino acids, loop structures with one intramolecular disulfide bond, and -sheet structures which are stabilised by two or three intramolecular disulfide bonds. Mammalian defensins are part of the last-mentioned group. The mammalian defensins can be subdivided into three main classes according to their structural differences: the -defensins, -defensins and the recently described -defensins. Mammalian -defensins are predominantly found in neutrophils and in small intestinal Paneth cells, whereas mammalian -defensins have been isolated from both leukocytes and epithelial cells. Recently, two novel human -defensins, human beta-defensin-3 (HBD-3), and human beta-defensin-4 (HBD-4) have been discovered. Similar to HBD-1 and HBD-2, HBD-3 has microbicidal activity towards the Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli) and the yeasts Candida albicans and Malassezia furfur. In addition, HBD-3 kills Gram-positive bacteria such as Streptococcus pyogenes or Staphylococcus aureus, including multi-resistant S. aureus strains, and even vancomycin-resistant Enterococcus faecium. In contrast to HBD-1 and HBD-2, significant expression of HBD-3 has been demonstrated in non-epithelial tissues, such as leukocytes, heart and skeletal muscle. HBD-4 is expressed in certain epithelia and in neutrophils. Its bactericidal activity against P. aeruginosa is stronger than that of the other known -defensins. Here we present an overview of human antimicrobial peptides with some emphasis on their antifungal properties.J.J. Schneider and A. Unholzer contributed equally to this work     

Multiple mechanisms govern the dynamics of depression at neocortical synapses of young rats

Synaptic transmission between pairs of excitatory neurones in layers V ( N = 38) or IV ( N = 6) of somatosensory cortex was examined in a parasagittal slice preparation obtained from young Wistar rats (14–18 days old). A combined experimental and theoretical approach reveals two characteristics of short-term synaptic depression. Firstly, as well as a release-dependent depression, there is a release-independent component that is evident in smaller postsynaptic responses even following failure to release transmitter. Secondly, recovery from depression is activity dependent and is faster at higher input frequencies. Frequency-dependent recovery is a Ca²⁺-dependent process and does not reflect an underlying augmentation. Frequency-dependent recovery and release-independent depression are correlated, such that at those connections with a large amount of release-independent depression, recovery from depression is faster. In addition, both are more pronounced in experiments performed at physiological temperatures. Simulations demonstrate that these homeostatic properties allow the transfer of rate information at all frequencies, essentially linearizing synaptic responses at high input frequencies.     

Chondroitin sulfate A released from platelets blocks RANTES presentation on cell surfaces and RANTES-dependent firm adhesion of leukocytes

The sequestration of chemokines on the surface of microvascular endothelium is an early event in the selective recruitment of leukocytes. The sequestration and presentation of chemokines must be tightly controlled to confine the extravasation of leukocytes and to prevent uncontrolled inflammation. We investigated whether soluble molecules released under physiological conditions could control chemokine immobilization on cell surfaces and function as regulatory chemokine binding molecules. We determined that human serum contains a molecule that suppresses RANTES (CCL5) binding to endothelial cells, PBMC and CHO cells. Using platelet-rich and platelet-free plasma, serum from patients with thrombocytopenia, and purified platelets, we identified platelets as the source of the chemokine-binding molecule and further identified it as chondroitin sulfate A. In contrast to platelet-derived fully-sulfated chondroitin sulfate A, low-sulfated chondroitin sulfate A present in plasma was almost inactive. Under physiological flow conditions chondroitin sulfate A was found to block RANTES-mediated firm adhesion of monocytes to endothelial cells. It also prevented RANTES-mediated influx of calcium in CCR5-transfected CHO cells while internalization of CCR5 was only marginally reduced. Taken together, chondroitin sulfate A released from platelets appears to act as an important regulatory molecule for cellular responses to chemokines.     

Viral hemagglutinin augments peptide-specific cytotoxic T cell responses

In attempt to increase the induction of peptide-specific cytolytic T cells (CTL) we investigated the effect of the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene product on the activation of peptide-specific CTL. Spleen cells of CH3 mice immunized against the influenza nucleoprotein peptide 50–63 (NP 50–63) were restimulated in vitro (i) with peptide-pulsed syngeneic fibroblast cells (Ltk^?) as antigen-presenting cells, which were in addition (ii) infected with NDV or (iii) stably transfected with the HN cDN A of NDV. A greater than sixfold increase in peptide-specific CTL responses was observed in cultures restimulated with peptide-pulsed Ltk^? cells which co-expressed viral hemagglutinin due to either infection or transfection. A similar augmentation was seen in CTL responses against other types of antigen (major histocompatibility complex alloantigens, minor histocompatibility antigens or tumor antigens) when suboptimal cultures were stimulated with the respective antigen-presenting cells modified by NDV infection. These findings suggest that NDV or viral HN expressed on antigen-presenting cells or tumor cells can exert a T cell co-stimulatory function.