Rupture of the gastrocnemius muscle at its distal musculotendinous junction: conservative treatment and outcomes in 11 dogs.

CASE HISTORY: Medical records from three veterinary referral centres and a university veterinary teaching hospital in Australia and the USA were reviewed to identify dogs with a diagnosis of distal gastrocnemius musculotendinous junction rupture (DGMJR) that were treated without surgery between 2007 and 2020. CLINICAL AND IMAGING FINDINGS: All dogs (n = 11) presented with unilateral, pelvic limb lameness and bruising, swelling or pain on palpation at the distal musculotendinous junction. The diagnosis was confirmed with ultrasound or MRI in six dogs; radiographs were used to excluded stifle and tarsus pathology in four dogs; and five dogs were diagnosed on physical examination findings. TREATMENT AND OUTCOME: All dogs were managed conservatively, either with complete confinement alone (n = 10; median 9 weeks), external coaptation alone (n = 1), or a combination of both (n = 4). Sporting dogs (n = 7) were completely confined (median 22 weeks) for longer periods than companion dogs (n = 3; median 5 weeks).A good to excellent outcome was achieved for all cases in this cohort. The seven sporting dogs achieved an excellent outcome; returning to their previous level of sport, with complete resolution of lameness and recovery of a normal tibiotarsal stance. The four companion dogs achieved a good outcome; returning to their previous level of activity but with persistently increased tibiotarsal standing angle compared to the contralateral limb. CLINICAL RELEVANCE: Conservative treatment represents a viable treatment option for dogs with a rupture of the gastrocnemius muscle at its distal musculotendinous junction.

Effect of doxycycline on contralateral canine cranial cruciate ligament rupture. A prospective randomized clinical trial in 69 dogs.

OBJECTIVE: To evaluate whether doxycycline administered to dogs with unilateral cranial cruciate ligament rupture (Uni-CCLR) would decrease the risk of contralateral-CCLR (Co-CCLR). To evaluate predictors for Co-CCLR survival. To evaluate if a predisposition of Labrador Retrievers to Co-CCLR exists when compared to other breeds. METHODS: In this prospective randomized controlled clinical trial, 69 client-owned dogs with Uni-CCLR were randomly assigned to a doxycycline (group-D: 7.5 mg/kg PO BID x 6 weeks) or non-doxycycline (group-ND: negative control). Medical and imaging data, time from Uni- to Co-CCLR and to follow-up were recorded. Statistics included chi-squared test, logistic regression, Kaplan-Meier survival analysis, log rank test, survival curves, and frailty model (p <0.05). RESULTS: This study included 32 dogs in group-D, and 37 dogs in group-ND. Median follow-up was 54.5 and 61 months, respectively. Contralateral CCLR occurred in 53.1% and 48.6% at medians of 20 and 11 months, respectively. Doxycycline did not significantly decrease the risk of Co-CCLR (p = 0.83). This risk was decreased by 14.2% with each year of age but increased with each increasing kilogram of body weight and each increasing degree of tibial plateau angle by 5.4% and 9.7%, respectively. Labrador Retrievers were not significantly predisposed (p = 0.37). CLINICAL SIGNIFICANCE: At the dose regimen investigated doxycycline does not decrease the risk for Co-CCLR.

Accuracy of three pre- and intra-operative measurement techniques for osteotomy positioning in the tibial plateau levelling procedure.

OBJECTIVES: To prospectively compare the accuracy of three preoperative measurement techniques in tibial plateau levelling osteotomy (TPLO) planning. METHODS: Fifty-nine dogs were randomly assigned to one of three measurement techniques; A, B or C. Surgeons measured the intended osteotomy location on preoperative radiographs according to the assigned technique. Measurements were used intra-operatively during osteotomy placement. Postoperative measurements were made by a single blinded observer and compared to preoperative measurements. RESULTS: Fifty-one dogs were included for final statistical analysis. The mean absolute differences between pre- and postoperative measurements was 1.72 mm ± 0.958, 1.79 mm ± 1.010, and 3.56 mm ± 1.839, for techniques A, B and C, respectively. No significant differences were identified for patient age, gender, limb or surgeon. Techniques A and B were not significantly different (p = 0.8799). Techniques A and B were significantly more accurate than C (p = 0.0001 and p = 0.0003, respectively). Weight was significantly different among the groups (p = 0.047) but the statistical results did not change when an adjustment was made for bodyweight (p = 0.4971, p <0.001 and p = 0.0007, respectively). CLINICAL SIGNIFICANCE: Preoperative measuring for planning a TPLO osteotomy is recommended. Techniques A and B in the current study were clinically practical and significantly more accurate compared to technique C.

Handling of the tibial muscle envelope in tibial plateau levelling osteotomy - to elevate or not? A clinical study of 40 dogs.

OBJECTIVE: To compare the outcome of the tibial plateau levelling osteotomy (TPLO) procedure, using a 6-hole 3.5 mm locking TPLO plate and performed with the muscle elevation technique (ET) and placement of sponges, to the TPLO without performing these steps (non-elevation-technique [NET]). MATERIAL AND METHODS: Medical records and radiographs of dogs with ET (n = 21) or NET (n = 19) were retrospectively reviewed. Signalment, TPLO procedure side, meniscal treatment, surgery time, haemorrhage, pre- and postoperative tibial plateau angle, assistant, amount of rehabilitation, bone healing (cortical, osteotomy, combined healing scores), complications, limb function, recovery time and follow-up were recorded and analysed using multivariate analysis. A value of p <0.05 was considered significant. RESULTS: Surgery time was significantly shorter with the NET (68.5 min ± 3.4) than with the ET (87.8 min ± 3.4) (p <0.01). No significant differences were detected for all other evaluated factors. Soft tissue trauma was minimal and none of the dogs suffered severe haemorrhage. The bone healing scores with the NET and the ET were not significantly different (p = 0.1, p = 0.2, p = 0.1). Complications were rare, minor and not significantly different between groups (p = 0.73). CLINICAL RELEVANCE: The results of this in vivo study indicate that NET is a feasible technique that can be considered for the clinical setting.

Minimally displaced tibial tuberosity avulsion fracture in nine skeletally immature large breed dogs.

OBJECTIVE: The objective of this study was to describe the clinical and radiographic features, as well as the treatment and outcome of minimally displaced tibial-tuberosity-avulsion-fractures (MDTTAF). MATERIALS AND METHODS: Signalment, history, diagnostics, therapy, and outcome were recorded. Follow-up was documented as re-examination, radiographic assessment or telephone conversation. RESULTS: Nine large breed dogs that were presented with lameness originating from the proximal tibia were included. All showed signs of pain when pressure was applied to the tibial tuberosity. There was no stifle instability or intra-articular disease. The main feature on mediolateral radiographs was a widened tibial-tuberosity-physis with reactive new bone and loss of edge definition of the epiphyseal and metaphyseal margins. Non-surgical treatment was chosen in eight dogs, and surgery in one dog. Radiographic follow-up showed progressive closure of the tibial-tuberosity-physis and healing. Clinical signs resolved at a median of 28 days (range: 14-120). DISCUSSION: Minimally displaced tibial-tuberosity-avulsion-fractures should be a differential diagnosis in skeletally immature large breed dogs older than nine months of age with signs of subtle pelvic-limb lameness, and signs of proximal tibial pain, but no evidence of stifle joint disease. Thorough clinical examination and critical review of bilateral radiographs are important to diagnose MDTTAF. The outcome in these cases suggests that the prognosis for MDTTAF is excellent. Age and size of the affected dogs in this study differ from an earlier publication that illustrated more severely displaced tibial tuberosity avulsion fractures, occurring mainly in terriers around five months of age.

Deformity secondary to bilateral incomplete ossification of the humeral condyle in a German Shorthaired Pointer dog. A case report with ten-year follow-up.

A five-month-old male, German Shorthaired Pointer dog was presented for severe, bilateral, thoracic-limb-lameness, with elbow swelling, pain, and crepitus. Radiography and computed tomography confirmed bilateral incomplete ossification of the humeral condyles (IOHC), with a non-displaced incomplete fracture of the left medial epicondylar crest and condylar deformity, characterised by enlargement of the trochleas with extension of the disto-medial aspect of the bone below the normal elbow joint level, and a deformed proximo-medial aspect of the ulna and radius. Transcondylar lag screws were placed bilaterally in an attempt to prevent fracture. Microscopic examination of biopsies, harvested from both humeral condyles, was supportive of IOHC. Six years after surgery, radiographs showed severe osteoarthritis and it appeared that fusion of the humeral condyles had not occurred. The dog followed an active life style until 10 years after surgery when the elbows showed almost no observable range-of-motion. However, discomfort was evident only after heavy exercise. Incomplete ossification of the humeral condyles may be associated with osteoarthritis, risk for non-union, implant failure, or secondary condylar fracture. This dog maintained good limb function despite these concerns, and despite bilateral humeral condylar deformity as well as development of severe osteoarthritis. This is the first report of a German Shorthaired Pointer dog with IOHC and the first histological description supportive of this condition in a five-month-old dog.

Does Osgood-Schlatter disease exist in the dog?

Osgood-Schlatter disease (OSD) is a condition affecting human adolescents in which there is partial separation of bone fragments from the tibial tuberosity at the site of insertion of the patellar ligament to the tibial tuberosity. Tensile trauma seems to be the most likely aetiology. Clinical signs in people consist of swelling and pain at the proximal part of the tibial tuberosity and around the distal end of patellar ligament. Radiographs frequently show small ossicles at the patellar ligament insertion. Conservative treatment is usually curative. The term OSD has also been used for the canine patient. However, radiographs of these patients typically show an enlarged radiolucent line at the apophyseal plate of the tibial tuberosity. This finding is consistent with a mild avulsion fracture of the canine tibial tuberosity. Based on the radiographic differences between the two species, it seems more appropriate to use the term OSD only for people. The purpose of this paper is to review the literature on OSD in people and the reports of injuries to the proximal tibial tuberosity in dogs. In addition, a new classification system for tibial tuberosity avulsion injuries in the immature dog is proposed, with an algorithm for management of this injury.

Mandibular and sublingual sialocoeles in the dog: a retrospective evaluation of 41 cases, using the ventral approach for treatment.

AIMS: To review results of the ventral approach for mandibular and sublingual sialoadenectomy for the treatment of sialocoeles associated with the mandibular and sublingual salivary glands in the dog, and to determine rates of recurrence and complication following this procedure. METHODS: Thirty-nine dogs with 41 sialocoeles that underwent surgical intervention were retrospectively evaluated with respect to signalment, aetiology, location of sialocoeles, duration of clinical signs, treatment prior to referral, post-operative use of antibiotics and drains, complications, and recurrence. RESULTS: The mean age at the time of surgery was 5.1 (SD 3.8) years, and duration of clinical signs 6.6 (SD 10.6) months. Long-term follow-up was available for 31 dogs; the minimum was 8 months and mean 47.7 (SD 25.8) months post-surgery. There was no recurrence of sialocoeles following the ventral approach for mandibular and sublingual sialoadenectomy. Postoperatively, 6/35 (17%) cases developed a seroma at the surgical site. No breed or sex predisposition was determined. The cause of the sialocoele was unknown in 36/41 (88%) cases. CONCLUSIONS AND CLINICAL RELEVANCE: Excellent clinical results were achieved with a low rate of complications using the ventral approach for mandibular and sublingual sialoadenectomy. The ventral approach is recommended to minimise the risk of recurrence of sialocoeles.

Platelet function in acute experimental pancreatitis induced by ischaemia-reperfusion.

BACKGROUND: Ischaemia-reperfusion (IR)-associated microcirculatory changes play a major role in acute post-transplantation pancreatitis. The pathophysiological role of platelets in these events is unknown. The aim of this study was to examine platelet adhesion and function during early reperfusion after pancreatic ischaemia. METHODS: Rats were subjected to warm pancreatic ischaemia by cross-clamping of the pancreatic vessels for 1 h. After 1 h of reperfusion, platelet-endothelium interaction was evaluated after platelet separation and staining by fluorescence microscopy. Amylase levels and pancreatic histology were evaluated 24 h after reperfusion. Animals treated according to an identical protocol, but without ischaemia, served as controls. RESULTS: Mild pancreatitis had developed by 24 h after IR; serum amylase levels were significantly higher than those in control animals. The numbers of adherent platelets in capillaries and venules were significantly increased, and platelet velocity in capillaries was significantly decreased, in the IR group compared with controls. There was significantly more oedema and inflammation in pancreatic tissue after IR. CONCLUSION: Warm ischaemia for 1 h followed by reperfusion for 24 h caused mild pancreatitis in this experimental model. The pancreatic microcirculation was characterized by pronounced platelet-endothelium interaction in capillaries and venules. These results suggest that platelet activation may play an important role in acute post-transplantation pancreatitis.

Activation of mitogen-activated protein kinases p42/44, p38, and stress-activated protein kinases in myelo-monocytic cells by Treponema lipoteichoic acid.

We have shown previously that phenol/water extracts derived from two novel Treponema species, Treponema maltophilum, and Treponema brennaborense, resembling lipoteichoic acid (LTA), induce cytokines in mononuclear cells. This response was lipopolysaccharide binding-protein (LBP)-dependent and involved Toll-like receptors (TLRs). Here we show that secretion of tumor necrosis factor-alpha induced by Treponema culture supernatants and extracted LTA was paralleled by an LBP-dependent phosphorylation of mitogen-activated protein kinases (MAPKs) p42 and p44, and p38, as well as the stress-activated protein kinases c-Jun N-terminal kinases 1 and 2. Phosphorylation of p42/44 correlated with an increase of activity, and tumor necrosis factor-alpha levels were significantly reduced by addition of inhibitors of p42/44 and p38, PD 98059 and SB 203580, respectively. Treponeme LTA differed from bacterial lipopolysaccharide regarding time course of p42/44 phosphorylation, exhibiting a prolonged activation of MAPKs. Furthermore, MAPK activation and cytokine induction failed to be strictly correlated. Involvement of TLR-4 for phosphorylation of p42/44 was shown employing the neutralizing anti-murine TLR-4 antibody MTS 510. In TLR-2-negative U373 cells, the compounds studied differed regarding MAPK activation with T. maltophilum leading to a stronger activation. In summary, the data presented here show that treponeme LTA are able to activate the MAPK and stress-activated protein kinase pathway involving LBP and TLR-4.

LPS-binding protein protects mice from septic shock caused by LPS or gram-negative bacteria.

LPS-binding protein (LBP) recognizes bacterial LPS and transfers it to CD14, thereby enhancing host cell stimulation, eventually resulting in pathogenic states such as septic shock. Recently, LBP also was shown to detoxify LPS by transferring LPS into HDL particles in vitro. Thus, the predominant in vivo function of LBP has remained unclear. To investigate the biological activity of acute phase concentrations of recombinant murine LBP, high concentrations of LBP were investigated in vitro and in vivo. Although addition of low concentrations of LBP to a murine macrophage cell line enhanced LPS-induced TNF-alpha synthesis, acute phase concentrations of LBP blocked this effect in comparison to low-dose LBP. When injected into mice intraperitoneally, LBP inhibited LPS-mediated cytokine release and prevented hepatic failure resulting in a significantly decreased mortality rate in LPS-challenged and D-galactosamine-sensitized mice, as well as in a murine model of bacteremia. These results complement a recent study revealing LBP-deficient mice to be dramatically more susceptible to an intraperitoneal Salmonella infection as compared with normal mice. We conclude that acute phase LBP has a protective effect against LPS and bacterial infection and may represent a physiologic defense mechanism against infection. Despite the limitations of any murine sepsis model, the results shown may imply that LBP could have beneficial effects during gram-negative peritonitis in humans.

Lipopolysaccharide and pneumococcal cell wall components activate the mitogen activated protein kinases (MAPK) erk-1, erk-2, and p38 in astrocytes.

Cell wall compounds of gram-positive bacteria are capable of inducing the biosynthesis of proinflammatory cytokines in CNS cells in a similar way as lipopolysaccharide (LPS) of gram-negative bacteria does. Astrocytes, which lack the CD14 LPS receptor, have also been shown to respond to LPS-stimulation by increased cytokine synthesis. However, almost nothing is known about signaling steps involved in this process. We have therefore examined signaling events in primary cultures of rat astrocytes and the human astrocytoma cell line U373MG, brought about by LPS and pneumococcal cell walls (PCW). Of particular interest to us was the tyrosine phosphorylation patterns and activation states of three members of the mitogen activated protein kinase (MAPK) family, i.e., extracellular signal-regulated protein kinase (erk)-1, erk-2, and the recently identified p38. We show that LPS and PCW initiate tyrosine phosphorylation and activation of erk-1, erk-2, and p38 in a dose-dependent fashion. Inhibitors of tyrosine phosphorylation were able to alleviate this effect and also blocked cytokine production of astrocytes. Both, LPS- and PCW-induced responses of astrocytic cells required the presence of soluble CD14 (sCD14) present in serum. Unraveling the signaling steps induced by bacterial compounds in cells of the CNS may potentially help to elucidate the pathomechanisms of meningitis and central nervous complications of sepsis and may offer options for novel treatment strategies.

Lipopolysaccharide activates caspase-1 (interleukin-1-converting enzyme) in cultured monocytic and endothelial cells.

Interleukin-1 beta (IL-1 beta) is a pleiotropic proinflammatory cytokine. Mechanisms leading to its secretion include not only release of newly synthesized protein, but also cleavage of a preformed immature precursor protein into an active secretory form by the intracellular protease caspase-1 (formerly termed IL-1-converting enzyme [ICE]). Caspase-1 belongs to a rapidly growing family of cysteine proteases with substrate specificity for aspartate involved in cellular apoptosis. We have used an assay determining the caspase-1 activity based on cleavage of a fluorogenic peptide substrate to elucidate its role in lipopolysaccharide (LPS)-induced secretion of IL-1 beta. We show that LPS induces moderate caspase-1 activity in the monocytic cell line THP-1, in freshly isolated peripheral blood monocytes, and in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent fashion. Caspase-1 activation by LPS was associated with cleavage of the IL-1 beta precursor protein that was followed by release of the mature IL-1 beta protein in monocytic cells. In contrast, subsequent release of IL-1 beta by HUVECs was not significant. LPS-induced caspase-1 activation appeared not to result from modulation of caspase-1 transcript accumulation and inhibition of caspase-1 activity was accomplished by two specific inhibitors, YVAD-CHO and YVAD-CMK, capable of alleviating the release of mature IL-1 beta. Taken together, these results show that LPS moderately activates caspase-1 and that caspase-1 activation contributes to LPS induction of IL-1 beta secretion.

Control of transcriptional activation of the lipopolysaccharide binding protein (LBP) gene by proinflammatory cytokines.

The lipopolysaccharide binding protein (BLP) is of major importance for endotoxin recognition, presentation and subsequent cytokine induction in immune cells. As a member of a growing family of structurally and functionally related proteins, LBP is synthesized in hepatocytes and constitutively secreted into the bloodstream. During the acute-phase response, however, LBP levels rise substantially. In this article the mechanisms of induction of LBP protein synthesis are highlighted. Induction of LBP in hepatocytes is the result of transcriptional and posttranscriptional mechanisms, as shown by nuclear run-on and RNA half-life experiments. Cloning of the 5' flanking region of the LBP gene gave results consistent with the LBP promoter as a typical acute-phase protein promoter. Reporter-gene assays employing the Luciferase gene and mutation variants of the LBP promoter revealed that integrity of a common acute-phase promoter motif, binding STAT-3, is essential for activation of the LBP promoter. Elucidating the transcriptional activation mechanism could show the way how to therapeutically lower LBP levels in high-risk patients in order to reduce their susceptibility to Gram-negative septic shock.

Similar organization of the lipopolysaccharide-binding protein (LBP) and phospholipid transfer protein (PLTP) genes suggests a common gene family of lipid-binding proteins.

The transfer of lipids in aqueous environments such as serum has been attributed to a recently characterized class of proteins. Abnormal regulation of serum lipids by these proteins is thought to be a key event in the pathophysiology of cardiovascular diseases. Lipopolysaccharide (endotoxin) binding protein (LBP) was identified by virtue of its ability to bind bacterial lipid A. We have analyzed the exon-intron organization of the LBP gene and the nucleotide sequence of its approximately 20 kb spanning 5'- and 3'-untranslated regions. When comparing the genomic organization of LBP with that of two other genes coding for lipid transfer proteins, significant homologies were found. The LBP gene includes 15 exons, and the 2-kb promoter contains recognition elements of acute phase-typical reactants and a repetitive 12-mer motif with an as yet unknown protein-binding property. Detailed sequence comparison revealed a closer relatedness of LBP with PLTP than with CETP as demonstrated by an almost identical intron positioning. This high degree of similarity supports functional studies by others suggesting that like LBP, PLTP may also be able to bind and transport bacterial lipopolysaccharide.

The transcriptional activation pattern of lipopolysaccharide binding protein (LBP) involving transcription factors AP-1 and C/EBP beta.

Lipopolysaccharide (LPS) Binding Protein (LBP) is an acute phase protein with the ability to recognize bacterial LPS and transport it to the CD14 molecule or into HDL particles. It is synthesized in hepatocytes and secreted into the blood stream. LBP levels significantly rise during the acute phase response and levels of LBP may be important for an appropriate host reaction to bacterial challenge and for developing the sepsis syndrome. In order to elucidate the mechanisms of LBP regulation we investigated its transcription pattern and performed promoter studies under experimental conditions mimicking an acute phase scenario. In human hepatoma cell lines stimulation with IL-1 beta, IL-6, TNF-alpha and dexamethasone leads to strong transcriptional activation of the LBP gene in a dose- and time-dependent manner. IL-6 alone induces LBP significantly, whereas IL-1 beta mainly increases the IL-6 effect when applied in combination. Our results furthermore show that AP-1 and C/EBP beta are transcription factors involved in the activation of the LBP gene, as revealed by Luciferase reporter gene analysis and electromobility shift assays. Elucidating the mechanism of transcriptional activation of LBP potentially may help in understanding host-pathogen response patterns and mechanisms involved in the acute phase reaction and in the pathophysiology of sepsis.

Effects of site-directed mutagenesis of basic residues (Arg 94, Lys 95, Lys 99) of lipopolysaccharide (LPS)-binding protein on binding and transfer of LPS and subsequent immune cell activation.

LPS-binding protein (LBP) is a 60-kDa acute phase glycoprotein capable of binding the LPS of Gram-negative bacteria and facilitating its diffusion. This process is thought to be of potential importance in inflammatory reactions and pathogenic states such as septic shock syndrome. Here, we report on the identification of a LPS binding domain within the LBP molecule and on the identification of single amino acids important for binding of LPS by LBP. Several synthetic LBP peptides inhibited LPS-LBP interaction, and amino acids Arg 94 and Lys 95 were centrally located in these inhibitory peptides. LBP mutants with amino acid exchanges within this region were expressed and tested in five different functional assays: binding to immobilized LPS; facilitation of binding of LPS aggregates to monocytes; transfer of LPS monomers from aggregates to soluble CD14; transfer of soluble CD14-bound LPS monomers to high density lipoprotein (HDL); and enhancement of LPS-induced cell activation. The double mutant Glu 94/Glu 95 was completely lacking LPS binding, transfer, and cell stimulatory activity, indicating that the integrity of amino acids 94 and 95 is required for LBP function. While mutations of amino acids Arg 94 or Lys 95 into alanine reduced the LPS binding activity of LBP dramatically, the ability to facilitate binding of LPS aggregates to membrane CD14 at the cell surface was retained. These findings emphasize the distinction between binding of LPS aggregates to cells, which is not associated with cell stimulation, and binding of LPS monomers to CD14, which leads to cell stimulation.

Functional expression and cellular mRNA localization of a G protein-activated K+ inward rectifier isolated from rat brain.

We have cloned by homology screening from a rat brain cDNA library a GIRK3-type (Kir 3.3) inwardly rectifying K+ channel subunit with high structural similarity to other subfamily members whose activity is thought to be controlled by receptor-stimulated G proteins. When heterologously expressed both in Xenopus oocytes and in mammalian COS-7 cells, rbGIRK3 subunits individually fail to form functional channels. In contrast, when coexpressed with other GIRK subunits, rbGIRK3 gives rise to prominent currents which are enhanced by the stimulation of coexpressed 5-HT1A receptors. In situ hybridizations show that of all GIRK subunits rbGIRK3 is most widely distributed and strongly expressed throughout the rat brain and thus may play an important role in central signal processing.

Lipopolysaccharide induces the rapid tyrosine phosphorylation of the mitogen-activated protein kinases erk-1 and p38 in cultured human vascular endothelial cells requiring the presence of soluble CD14.

Human vascular endothelial cells (HUVECs), which do not display the lipopolysaccharide (LPS) receptor CD14, were examined for protein tyrosine phosphorylation after LPS stimulation in the presence and absence of soluble CD14 (sCD14). By phosphotyrosine Western blotting and immunocomplex kinase assays we show that LPS was capable of inducing in these cells rapid protein tyrosine phosphorylation and kinase activation of two members of the mitogen-activated protein kinase (MAPK) family erk-1 and the newly discovered p38, requiring the presence of sCD14. LPS-induced tyrosine phosphorylation of MAPK was associated with increased transcript- and surface protein expression of intracellular adhesion molecule-1 by HUVECs. MAPK phosphorylation and activation was induced by LPS in concentrations as little as 30 ng/mL and as early as 15 minutes after stimulation. Furthermore, tyrosine kinase inhibitors such as Genistein partially inhibited this effect. These results show that LPS triggers similar signaling events in both CD14+ myelo-monocytic cells and cells lacking the putative LPS-receptor CD14, suggesting the presence of a common, yet unidentified element in LPS-signaling in both cell types.

Gene transfer of human TNF alpha into glioblastoma cells permits modulation of mdr1 expression and potentiation of chemosensitivity.

Despite substantial advances in the surgery, radiotherapy and chemotherapy of gliomas, the prognosis of patients with glioblastomas has still not improved. Disappointing results in chemotherapy of glioblastomas resulting from multi-drug resistance (MDR) prompted us to investigate the influence of cytokine gene transfer in glioblastoma cells on the expression of P-glycoprotein and on chemosensitivity of transduced cells. Several investigations have shown that malignant gliomas express P-glycoprotein at high levels. The P-glycoprotein is a product of the multi-drug resistance gene (mdr1) and functions as an energy-dependent efflux pump which decreases drug accumulation and cytotoxicity. Since tumour necrosis factor alpha (TNF alpha) is a powerful anti-cancer agent used in clinical trials and gene therapy protocols, this cytokine gene was chosen for the present investigations. Transduction of the human TNF alpha (hTNF) gene carrying retroviral vector pN2tk-hTNF into U373MG human glioblastoma cells resulted in expression and secretion of biologically active hTNF. Release of transduced hTNF reduces P-glycoprotein expression and is associated with enhanced rhodamine-123 uptake and potentiation of cytotoxicity of the MDR-relevant drugs vincristine and doxorubicin. Furthermore, the transfected cell clones showed a reduced growth rate compared to the parental cells.