Tamás Józsa

High-throughput live-cell imaging reveals differential inhibition of tumor cell proliferation by human fibroblasts

Increasing evidence indicates that cancer development requires changes both in the precancerous cells and in their microenvironment. To study one aspect of the microenvironmental control, we departed from Michael Stokers observation (Stroker et al, J Cell Sci 1966;1:297–310) that normal fibroblasts can inhibit the growth of admixed cancer cells (neighbour suppression). We have developed a high‐throughput microscopy and image analysis system permitting the examination of live mixed cell cultures growing on 384‐well plates, at the single cell level and over time. We have tested the effect of 107 samples of low passage number (<5) primary human fibroblasts from pediatric and adult donors, on the growth of six human tumor cell lines. Three of the lines were derived from prostate carcinomas, two from lung carcinomas and one was an EBV‐transformed lymphoblastoid line. Labeled tumor cells were grown in the presence of unlabeled fibroblasts. The majority of the tested fibroblasts inhibited the proliferation of the tumor cells, compared to the control cultures where labeled tumor cells were co‐cultured with unlabeled tumor cells. The proliferation inhibiting effect of the fibroblasts differed depending on their site of origin and the age of the donor. Inhibition required direct cell contact. Mouse 3T3 fibroblasts inhibited the growth of SV40‐transformed 3T3 cells and human tumor cells, showing that the inhibitory effect could prevail across the species barrier. Our high‐throughput system allows the quantitative analysis of the inhibitory effect of fibroblasts on the population level and the exploration of differences depending on the source of the normal cells.

Leptin receptor deficient diabetic (db/db) mice are compromised in postnatal bone regeneration

Increased fragility fracture risk with improper healing is a frequent and severe complication of insulin resistance (IR). The mechanisms impairing bone health in IR are still not fully appreciated, which gives importance to studies on bone pathologies in animal models of diabetes. Mice deficient in leptin signaling are widely used models of IR and its comorbidities. Leptin was first recognized as a hormone, regulating appetite and energy balance; however, recent studies have expanded its role showing that leptin is a link between insulin-dependent metabolism and bone homeostasis. In the light of these findings, it is intriguing to consider the role of leptin resistance in bone regeneration. In this study, we show that obese diabetic mice lacking leptin receptor (db/db) are deficient in postnatal regenerative osteogenesis. We apply an ectopic osteogenesis and a fracture healing model, both showing that db/db mice display compromised bone acquisition and regeneration capacity. The underlying mechanisms include delayed periosteal mesenchymatic osteogenesis, premature apoptosis of the cartilage callus and impaired microvascular invasion of the healing tissue. Our study supports the use of the db/db mouse as a model of IR associated bone-healing deficits and can aid further studies of mesenchymatic cell homing and differentiation, microvascular invasion, cartilage to bone transition and callus remodeling in diabetic fracture healing.

Atresia of the ileocecal junction with agenesis of the ileocecal valve and vermiform appendix: report of a case.

Intestinal atresia involving the ileocecal region is a very rare intestinal malformation, and the presence or absence of the ileocecal valve influences its surgical management. We report the case of a male newborn with a provisional diagnosis of distal ileal atresia, in whom laparotomy revealed that the entire ileocecal region was atretic with an absent ileocecal valve and appendix vermiformis. We resected the dilated terminal ileum together with the atretic segment and performed an ileocolic anastomosis between the terminal ileum and the transverse microcolon without valve reconstruction. When last seen, 8 months after the operation, the baby was developing normally. Ileocolic anastomosis without valve replacement appears to be sufficient if an ileocecal valve is completely absent and only a short segment of the terminal ileum is lost.

Hypothermia translocates nitric oxide synthase from cytosol to membrane in snail neurons

Neuronal nitric oxide (NO) levels are modulated through the control of catalytic activity of NO synthase (NOS). Although signals limiting excess NO synthesis are being extensively studied in the vertebrate nervous system, our knowledge is rather limited on the control of NOS in neurons of invertebrates. We have previously reported a transient inactivation of NOS in hibernating snails. In the present study, we aimed to understand the mechanism leading to blocked NO production during hypothermic periods of Helix pomatia. We have found that hypothermic challenge translocated NOS from the cytosol to the perinuclear endoplasmic reticulum, and that this cytosol to membrane trafficking was essential for inhibition of NO synthesis. Cold stress also downregulated NOS mRNA levels in snail neurons, although the amount of NOS protein remained unaffected in response to hypothermia. Our studies with cultured neurons and glia cells revealed that glia–neuron signaling may inhibit membrane binding and inactivation of NOS. We provide evidence that hypothermia keeps NO synthesis “hibernated” through subcellular redistribution of NOS.

Follow-up of thrombin generation after prostate cancer surgery: global test for increased hypercoagulability.

Recent studies provided evidence that evaluation of thrombin generation identifies patients at thrombotic risk. Thrombin generation has a central role in hemorrhage control and vascular occlusion and its measurement provides new metrics of these processes providing sufficient evaluation of an individual’s hemostatic competence and response to anticoagulant therapy. The objective of the study is to assess a new measure of hypercoagulability that predisposes to venous thromboembolism in the postoperative period after radical prostatectomy. Pre- (day-1) and postoperative (hour 1, day 6, month 1 and 10) blood samples of 24 patients were tested for plasma thrombin generation (peak thrombin), routine hematology and hemostasis. Patients received low molecular weight heparin for thromboprophylaxis. Peak thrombin levels were higher in patients compared to controls at baseline (p<0.001), and elevated further in the early postoperative period (p<0.001). Longer general anesthesia and high body mass index were associated with increased thrombin generation after surgery (p = 0.024 and p = 0.040). D dimer and fibrinogen levels were higher after radical prostatectomy (p = 0.001 and p<0.001). Conventional clotting tests remained within the reference range. Our study contributed to the cognition of the hypercoagulable state in cancer patients undergoing pelvic surgery and revealed the course of thrombin generation after radical prostatectomy. Whilst it is unsurprising that thrombin generation increases after tissue trauma, further evaluation of this condition during the postoperative period would lead urologists to an international and well-supported consensus regarding thromboprophylaxis in order to provide better clinical outcome. Considering the routine evaluation of procoagulant activity and extending prophylactic anticoagulant therapy accordingly may potentially prevent late thrombotic events.

Acetylcholine inhibits nitric oxide (NO) synthesis in the gastropod nervous system

Acetylcholine (ACh) is one of the main signals regulating nitric oxide synthase (NOS) expression and nitric oxide (NO) biosynthesis in mammals. However, few comparative studies have been performed on the role of ACh on NOS activity in non-mammalian animals. We have therefore studied the cholinergic control of NOS in the snail Helix pomatia and compared the effects of ACh on NO synthesis in the enteric nervous system of the snail and rat. Analyses by the NADPH-diaphorase reaction, immunocytochemistry, purification with ion-exchange chromatography, Western-blot, and quantitative polymerase chain reaction have revealed the expression of neuronal NOS in the rat intestine and of a 60-kDa subunit of NOS in the enteric nerve plexus of H. pomatia. In H. pomatia, quantification of the NO-derived nitrite ions has established that NO formation is confined to the NOS-containing midintestine. Nitrite production can be elevated by L-arginine but inhibited by Nω-nitro-L-arginine. In rats, ACh moderately elevates nitrite production, whereas ACh, the nicotinic receptor agonists (nicotine, acetyl thiocholine iodide, metacholine) and the cholinesterase inhibitor eserine reduce enteric nitrite formation in snails. The nicotinic receptor antagonist tubocurarine also provokes nitrite liberation, whereas the muscarinic receptor agonists or antagonists have no significant effect in snails. In the presence of EDTA or tetrodotoxin, ACh fails to inhibit nitrite production. In pharmacological studies, we have found that ACh contracts the midintestinal muscles and, in snails, simultaneously reduces the antagonistic muscle relaxant effect of L-arginine. Our experiments provide the first evidence for an inhibitory regulation of neuronal NO synthesis by ACh in an invertebrate species.

Effect of hydrocele on appendix testis in children.

The purpose of this study was to investigate the effects of an elevated hydrostatic pressure of hydrocele on the structural integrity and steroid receptor expression pattern of the appendix testis in children. Twenty-six testicular appendages were obtained from boys (aged between 13 and 79 months, mean 40 months) who underwent surgical exploration because of hydrocele or congenital inguinal hernia. The tissue sections of testicular appendages were stained with hematoxylin-eosin. Immunohistochemistry and immunofluorescence laser microscopy were performed using monoclonal mouse anti-human receptors against androgen and estrogen receptors. Patients were divided into three groups: group A (n = 8) represented patients with groin hernia without hydrocele, who served as control group; group B (n = 7) represented patients with communicating hydrocele; and group C (n = 11) represented patients with noncommunicating hydrocele. The tissue sections of appendix testis expressed both androgen and estrogen receptors in all patients in groups A and B, and epithelial destruction was not present. The presence of androgen receptor (two of 11, P < 0.001) and estrogen receptor (four of 11, P = 0.006) was lower and the number of appendix testes with epithelial destruction was higher (eight of 11, P = 0.001) in group C. We demonstrated that groin hernia and communicating hydrocele did not influence the receptor expression pattern and the anatomic structure of testicular appendages, whereas noncommunicating hydrocele caused damage as indicated by the absence of steroid receptors and destruction of the epithelial surface. A better understanding of the physiological role of testicular appendages may change the indications of surgical treatment in patients with noncommunicating hydrocele.

Decreased Incidence of Appendix Testis in Cryptorchidism with Intraoperative Survey

Objective: Several authors have investigated the background of the process of testicular descent, but the role of the appendix testis has not been studied. The human appendix testis was found to express both estrogen and androgen receptors. We determined and compared the occurrence of testicular appendices intraoperatively in descended and undescended testes. Methods: The number of appendix testis was evaluated retrospectively in 208 boys who underwent uni- or bilateral orchiopexy, hydrocele or hernia repair and the testis was visible during operation. Results: The incidence of appendix testis was 76% (78 in 103) in descended and 24% (30 in 125) in undescended testes. Mean age at orchiopexy was lower in patients without appendix testis (39 months) compared to those patients who were found with appendix (61 months). Conclusion: The incidence of appendix testis was significantly lower (p < 0.05) in undescended testes, suggesting that the appendix testis might play a role in the process of testicular descent.

Hydrostatic Characteristics of the Ileocolic Valve and Intussuscepted Nipple Valves: An Animal Model

The ileocolic valve, in the dog, decelerates the passage of stools and prevents fecal reflux. A loss of anterograde resistance worsens the symptoms of short bowel syndrome. The absence of fecal reflux control enhances the risk of recurrence of Crohns disease. The aim of the present study was to examine what length of intussusception-like nipple valve (INV) should be constructed in order to restore the hydrostatic characteristics of the normal ileocecal valve. The anterograde and retrograde hydrostatic resistances of INVs of different lengths (4, 5, 6, or 7 cm) were compared with those of the normal ileocolic valve by using a contrast enema and x-ray monitoring in a canine model. It was found that the 4-cm-long INV may be sufficient to achieve an appropriate antireflux efficacy (59.60 ± 4.26 cm H2O) versus the ileocolic valve (25.80 ± 4.92 cm H2O), but this does not furnish an anterograde resistance comparable to that of the normal ileocolic valve (10.70 ± 1.15 cm H2O vs. 21.60 ± 3.96 cm H2O). We found that the appropriate length of the INV with which the anterograde resistance of the ileocolic valve could be attained in our model was between 6 and 7 cm. Thus, the shortest possible constructed INV should be effective in clinical conditions such as Crohns disease, but the recommended length in short bowel syndrome should be greater than this.

Expression of anti-Mullerian hormone receptor on the appendix testis in connection with urological disorders

The female internal sex organs develop from the paramesonephric (Mullerian) duct. In male embryos, the regression of the Mullerian duct is caused by the anti-Mullerian hormone (AMH), which plays an important role in the process of testicular descent. The physiological remnant of the Mullerian duct in males is the appendix testis (AT). In our previous study, we presented evidence for the decreased incidence of AT in cryptorchidism with intraoperative surgery. In this report, the expression of the anti-Mullerian hormone receptor type 2 (AMHR2), the specific receptor of AMH, on the AT was investigated in connection with different urological disorders, such as hernia inguinalis, torsion of AT, cysta epididymis, varicocele, hydrocele testis and various forms of undescended testis. The correlation between the age of the patients and the expression of the AMHR2 was also examined. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the receptors mRNA and protein levels, respectively. We demonstrate that AMHR2 is expressed in the ATs. Additionally, the presence of this receptor was proven at the mRNA and protein levels. The expression pattern of the receptor correlated with neither the examined urological disorders nor the age of the patients; therefore, the function of the AT remains obscure.