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Chronic inflammatory demyelinating polyradiculopathy (CIDP) and multifocal motor neuropathy (MMN) are seen as distinct entities with marked differences in pathophysiology and clinical, laboratory, and imaging features. We report a patient with an immune-mediated neuropathy in the borderland of CIDP and MMN, whose magnetic resonance imaging and cerebrospinal fluid (CSF) features strongly resembled CIDP, while the clinical course and treatment response suggested the diagnosis of MMN without conduction blocks. There is strong evidence that MMN is not a variant of CIDP and that these conditions can be separated pathologically. Our case report widens the spectrum of MMN presentations, indicating the existence of a clinical overlap syndrome of MMN and CIDP, and emphasizing the need for more precise criteria regarding CSF and nerve root imaging abnormalities in the differentiation of chronic immune-mediated neuropathies.

Pasteurella multocida is an important respiratory tract pathogen in intensive livestock farming, especially in pigs. Antimicrobial agents are frequently used to combat infections caused by this pathogen. In a study on antimicrobial resistance among respiratory tract pathogens of pigs from 30 German pig-producing farms, P. multocida isolates (n = 9) with high minimal inhibitory concentration (MIC) values of 16/304 mg/L (n = 2), 32/608 mg/L (n = 3) or ≥64/1216 mg/L (n = 4) for trimethoprim/sulfamethoxazole (1:19) and of ≥512 mg/L (n = 9) for trimethoprim (TMP) were detected in three of these farms. The genetic relatedness of the isolates was investigated via capsule-specific PCR and macrorestriction analyses with ApaI and SmaI. Pulsed-field gel electrophoresis revealed indistinguishable restriction patterns per farm, with slight differences between the three farms. All isolates represented capsular type A. Four representative isolates, that were subjected to whole genome sequencing, shared the multi-locus sequence type (ST) 3. Their plasmids were transformed into E. coli TOP10 with subsequent selection on TMP-containing agar plates. Antimicrobial susceptibility testing and plasmid analysis of the transformants confirmed that they were resistant to sulfonamides and trimethoprim and carried only a single small plasmid. This plasmid was completely sequenced and revealed a size of 6050 bp. Sequence analyses identified the presence of a resistance gene cluster comprising the genes sul2-ΔstrA-dfrA14-ΔstrA-ΔstrB. Further analysis identified a dfrA14 gene cassette being integrated into the strA reading frame. Neither the gene dfrA14 nor this gene cluster have been detected before in P. multocida.

An important approach in tumor therapy is combining substances with different action mechanisms aiming to enhance the antineoplastic effect, decrease the therapeutic dosage, and avoid resistance mechanisms. Moreover, evaluating compounds already approved for the treatment of non-neoplastic diseases is promising for new antineoplastic therapies. Sodium dichloroacetate (DCA) reactivates oxidative phosphorylation in the cancer cell mitochondria, reducing apoptosis resistance in cancer cells. Furthermore, metformin inhibits the proliferation of tumor cells and CD133+ cancer -stem-like cells. In the present study, we evaluated the independent and synergistic effect of metformin and DCA on the metabolic activity, cell proliferation, and apoptosis of a canine prostate adenocarcinoma (Adcarc1258) and a transitional cell carcinoma cell line (TCC1506) in comparison to a primary canine fibroblast culture. Determining metformin uptake in tumor cells was performed by quantitative HPLC. Depending on the dosage, metformin as a single agent inhibited the metabolic activity and cell proliferation of the tumor cells, showing only minor effects on the fibroblasts. Furthermore, 1 mM metformin increased apoptosis over 96 h in the tumor cell lines but not in fibroblasts. Additionally, metformin uptake into the tumor cells in vitro was measurable by quantitative HPLC. Synergistic effects for the combination therapy were observed in both neoplastic cell lines as well as in the fibroblasts. Based on these results, metformin might be a promising therapeutic agent for canine urogenital tumors. Further studies on kinetics, toxicology, bioavailability, and application of metformin in dogs are necessary.

Background and objectives Multifocal motor neuropathy (MMN) is a rare neuropathy and detailed descriptions of larger patient cohorts are scarce. The objective of this study was to evaluate epidemiological, clinical, and laboratory features of MMN patients and their response to treatment in Austria and to compare these data with those from the literature. Methods Anonymized demographic and clinical data about MMN patients until 31.12.2017 were collected from registered Austrian neurologists. Exploratory statistics on clinical and laboratory features as well as treatment regimens and responses were performed. Results 57 Patients with MMN were identified, resulting in a prevalence of 0.65/100.000. Mean age of onset was 44.1 ± 13.1 years, the diagnostic delay 5.5 ± 8.4 years. In 77% of patients, symptom onset was in the upper limbs, and in 92%, it occurred in distal muscles. Proximal onset was never observed in the lower limbs. At the final follow-up, the majority of patients had atrophy (88%) in affected regions. Definite motor conduction blocks (CB) were found in 54 patients. Anti-GM1-IgM antibodies were present in 43%. Treatment with intravenous immunoglobulins improved muscle strength and INCAT score initially, but at last follow-up, both scores deteriorated to values before treatment. Discussion The findings of the present study corroborate the previous findings in MMN. Onset typically occurs in the upper limbs and mostly distal, CBs are found in the majority of cases, while anti-GM1-IgM antibodies are detected in only approximately 40%. Our study underlines that the initial good response to treatment fades over time.

Background: Dialysis and kidney transplant patients are vulnerable populations for COVID-19 related disease and mortality. Methods: We conducted a prospective study exploring the eight week time course of specific cellular (interferon-I³ release assay and flow cytometry) or/and humoral immune responses (ELISA) to SARS-CoV-2 boost vaccination in more than 3100 participants including medical personnel, dialysis patients and kidney transplant recipients using mRNA vaccines BNT162b2 or mRNA-1273. Results: SARS-CoV-2-vaccination induced seroconversion efficacy in dialysis patients was similar to medical personnel (> 95%), but markedly impaired in kidney transplant recipients (42%). T-cellular immunity largely mimicked humoral results. Major risk factors of seroconversion failure were immunosuppressive drug number and type (belatacept, MMF-MPA, calcineurin-inhibitors) as well as vaccine type (BNT162b2 mRNA). Seroconversion rates induced by mRNA-1273 compared to BNT162b2 vaccine were 97% to 88% (p

Most patients with myasthenia gravis (MG) need long-term immunosuppressive therapy. However, conventional agents may have intolerable side effects, take too long or fail to achieve disease control. Rituximab (RTX) has emerged as an off-label treatment for refractory MG, but data on its use are still sparse. We conducted a retrospective nationwide study contacting all Austrian neurologists to provide anonymized data of all adult MG patients treated with RTX and minimum follow-up of 3 months. The Myasthenia Gravis Foundation of America Postintervention Status scale was used to assess outcomes. 34 (60.7%) of a total of 56 patients were women. Median (IQR) age at diagnosis of MG and start of RTX were 41.5 (24.3; 65.8) and 47.5 (33; 71) years, respectively. Antibodies (ab) against acetylcholine receptor (AchR) and muscle-specific tyrosine kinase (MuSK) were present in 69.6% and 25% of patients, respectively (seronegative: 5.4%). Before RTX, 47 (83.9%) patients had had plasma exchange, immune adsorption or immunoglobulins. Three months after RTX, 14 of 53 (26.4%) patients were in remission. At last follow-up after a median of 20 (10; 53) months, remission was present in 42.9% of patients and another 25% had minimal manifestations. Remission was more frequent in patients with MuSK ab vs. those with AchR ab (71.4% vs. 35.9%, p = 0.022). RTX was safe. The presence of MuSK ab independently predicted remission after RTX. In this retrospective study on RTX for MG, the largest to date, RTX appeared safe, efficacious and fast acting. Benefit from RTX was greatest in MuSK ab + MG.

Objective The recently developed bioactive glass PC-XG3, which is suitable to coat zirconia implant surfaces with high adhesion strength may reduce the time of osseointegration and the marginal bone loss following implantation. The glass composition has been previously evaluated for cytotoxicity on fibroblast cells, and will now be used to evaluate the cell behavior of osteoblast cells. Methods Three different surface morphologies were created with PC-XG3 on zirconia discs. A clinically tested zirconia implant surface as well as polished and machined zirconia served as a reference. Cell viability after 24 h, cell spreading after 30 min and 24 h and the respective morphology of human osteoblasts using scanning electron microscopy were evaluated. Additionally, the corrosive process of PC-XG3 in cell culture medium up to 7 d was measured. Results Initial cell behavior of human osteoblasts was not accelerated by the PC-XG3 surface when compared to zirconia. Additionally, it was found that a decreased surface roughness promoted initial cell spreading. Storage in cell culture medium resulted in the accumulation of C and N on the bioglass surface while Mg, Si, K and Ca were decreased and crack formation was observed. Significance Since initial spreading quality to a biomaterial is a crucial factor that will determine the subsequent cell function, proliferation, differentiation, and viability it can be assumed that a coating of zirconia implants with this bioactive glass will unlikely reduce osseointegration time.

The collaboration project VASIB aims at reducing the antibiotic consumption in pig production by integrating information from consulting expertise in clinical inspection, hygiene, epidemiology, microbiology and pharmacology. In this VASIB subproject, we investigated the antimicrobial susceptibility and relatedness of porcine respiratory tract pathogens. Bordetella bronchiseptica (n = 47), Pasteurella multocida (n = 18) and Streptococcus suis (n = 58) were obtained from weaner pigs at two farms. Antimicrobial susceptibility testing was performed by broth microdilution according to CLSI standards. Resistance genes were detected via specific PCR assays. Macrorestriction analysis was conducted to determine the relatedness of the isolates and to identify clones. The B. bronchiseptica isolates showed indistinguishable (farm 1) or two closely related XbaI-patterns (farm 2). Different SmaI-PFGE patterns of P. multocida isolates were obtained at three different time points. In contrast, PFGE analysis of S. suis indicated more than one fragment pattern per pig and time point. Isolates exhibiting indistinguishable PFGE patterns were considered to represent the same clone. This study showed that only two closely related B. bronchiseptica clones were present in both farms, which had low MICs to all antimicrobials, except to β-lactams. Different P. multocida clones were present at the three time points. They showed overall low MIC values, with two clones being resistant and one intermediate to tetracycline. S. suis clones were resistant to tetracycline (n = 19) and/or erythromycin/clindamycin (n = 16). They harboured the tetracycline resistance genes tet(O), tet(M) or tet(L) and/or the macrolide/lincosamide/streptogramin B resistance gene erm(B). Five penicillin-resistant S. suis clones were also detected.

Leptospirosis is a ubiquitous and potentially fatal zoonosis with protean manifestations. Human infection commonly occurs through contact with contaminated water or soil. In developed countries, leisure or household activities are increasingly associated with the disease. Within few months, we encountered five unrelated and autochthonous cases of severe leptospirosis, three of them requiring interim dialysis. In this case series, we present their clinical course. Furthermore, we provide an overview on the spectrum of organ involvement, with an emphasis on kidney injury, and comment on pitfalls in establishing the diagnosis. The considerable variance in presentation-with admissions both to internal and neurological units-emphasises the high index of suspicion required to arrive at the right diagnosis, particularly in countries of perceived low risk such as Austria.

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix®, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell–extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix®, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.

We read with interest the case report by Witczak et al .1 The authors describe a young woman who presented with headache, neck stiffness and confusion and was eventually found to have primary adrenal insufficiency. The authors speculate that the patient’s neurological manifestation was a consequence of the underlying adrenal failure, although they acknowledge that there is no reported association of the patient's initial presentation with adrenal crisis. We wonder whether a better recognized entity was considered by the authors, which could easily account for the patient's …

BACKGROUND The etiology of tumors arising in the biliary tract remains unclear. Several previous studies have detected Helicobacter pylori organisms in bile from patients with gallstones or cholecystitis. The objective of this study was to determine whether there is an association between H. pylori in bile and biliary tract carcinoma. METHODS The authors used polymerase chain reaction (PCR) assays to detect the presence of H. pylori in the stomach and bile from 89 patients: Sixty-three disease free patients had biliary calculi, 15 patients had carcinoma of the biliary tract, and 11 patients had neither gallstones nor carcinoma. Bile was considered to contain H. pylori only if the results of PCR determinations were positive in two or more samples assayed independently in two separate laboratories. RESULTS There was a strong association between the presence of H. pylori in the stomach and in the bile (P ≤ 0.01). Biliary H. pylori was associated with age but not with gender, and it was associated strongly with the clinical diagnosis. Patients with gallstones were 3.5 times as likely to have H. pylori in the bile compared with patients in a control group (95% confidence interval [95%CI], 0.8–15.8; P = 0.100), and H. pylori was 9.9 times more frequent in patients with biliary tract carcinoma compared with patients in the control group (95%CI, 1.4–70.5; P = 0.022). CONCLUSIONS There is a strong association between biliary tract carcinoma and H. pylori in bile. If these results are confirmed by prospective studies, H. pylori may be responsible for a significant proportion of malignant biliary tract disease. Cancer 2002;95:1946–53. © 2002 American Cancer Society. DOI 10.1002/cncr.10893

Coping with illness and the process of dying can be interpreted as an antinomy for a physician's work from ancient times. Successful coping is targeted by definition--at least implicitly--on preserving human life at the best quality possible. On the contrary, the process of dying leading to an individual's death remains out of scope. The aim of our study is to describe present concepts concerning the process of dying and successful coping with illness (i.e., with cancer) in order to look for the specific place of competent medical attendance and terminal care within this context.

Programmed cell death via the Fas receptor/Fas Ligand and DR4, DR5/TRAIL plays a major role in tumor escape and elimination mechanisms. It also promises to be an effective therapy alternative for aggressive tumors, as has been recently shown for colon, breast, and lung cancer cells. We attempted to clarify the role of these molecules in aggressivity of pancreatic carcinomas and to identify possible pathways as targets for therapy.Five pancreatic cell lines were investigated for the expression of FasL/Fas, DcR3, DR4, DR5/TRAIL, DcR1, DcR2, and other death pathways related molecules such as Bax, bcl-xL, bcl-2, FADD, and caspase-3 by flow cytometry, immunoblotting, and RT/PCR, both semiquantitative and real time (TaqMan). The susceptibility of these cell lines to apoptosis mediated by recombinant TRAIL was investigated. The effect of therapeutic agents (gemcitabine) on their susceptibility to TRAIL induced apoptosis was studied as well.Pancreatic adenocarcinomas expressed high levels of apoptosis-inducing receptors and ligands. They showed differential susceptibility to cell death induced by TRAIL, despite expressing intact receptors and signaling machineries. Treatment with commonly used therapeutic agents did not augment their susceptibility to apoptosis. This could be explained by the fact that they expressed differentially high levels of decoy receptors, as well as molecules known as inhibitors of apoptosis.The data suggest that pancreatic carcinoma cells have developed different mechanisms to evade the immune system. One is the expression of nonfunctional receptors, decoy receptors, and molecules that block cell death, such as bcl2 and bcl-xL. The second is the expression of apoptosis-inducing ligands, such as TRAIL, that could induce cell death of immune cells. The success in treating malignant tumors by recombinant TRAIL might apply to some but not all pancreatic tumors because of their differential resistance to TRAIL-induced cell death.

Background The diagnosis of biliary disease, namely malignant disorders, is frequently hampered by the inconclusive cytology. We investigated prospectively the frequency of molecular changes in p53 and ras compared with cytology in patients with primary or secondary hepato-biliary disease. Material and methods We investigated 118 consecutive patients, aged 24-89 with the following clincal diagnoses: choledocho/cholecystolithiasis (28), cholangiocellular carcinoma (21), gall bladder tumor (8), liver metastasis (3), autoimmune disease (8), chronic pancreatitis (16), pancreatic carcinoma (11), papillary disease (4), hepatic cirrhosis (6), cholangitis (2), anomalies (2), and normal (9). Bile was aspirated during routine endoscopic retrograde cholangio pancreatography (ERCP) or percutaneous transhepatic cholangiography (PTC). DNA was prepared freshly from a native aliquot, p53 mutations were detected by polymerase chain reaction (PCR) for exons 5 through 8 followed by TGGE. PCR for ras mutations was performed as RFLP-PCR with sequencing. Results In four cases, mutations in p53 could be found in exons 6 and 7. Twenty-two samples showed ras mutations; ras mutations were found in choledocholithiasis (4/28), bile duct (5/21), gall bladder (3/8) and pancreatic (1/11) carcinoma, liver metastasis (3/3), ulcerative colitis (2/3), PSC (1/2), and chronic pancreatitis (1/16). Cytology was clearly positive in seven cases, suspicious in three other, inconclusive in six, and negative in the rest. The molecular analysis resulted in a sensitivity of 33% and specificity of 87%, respectively, for the diagnosis of a malignant condition. Conclusion PCR for p53 and ras mutations may aid the diagnosis of primary and secondary (metastatic) hepatobiliary disease if a malignant condition of the bile ducts and the liver is suspected and cytology is inconclusive or negative. However, the incidence of p53 and ras mutations in bile seems less frequent than in other malignant conditions of the gastrointestinal tract and the pancreas and lower than in tissue, leaving a poor sensitiviy and specificity. Nevertheless, the presence of a p53 and/or ras mutation per se supports a clinical suspicion of malignancy, even when the conventional cytology is negative or inconclusive.

The prognosis of pancreatic cancer is poor, and current medical treatment is mostly ineffective. The aim of this study was to design a new treatment modality in an animal model system. We describe here a novel treatment strategy employing a mouse model system for pancreatic carcinoma. Embryonal kidney epithelial cells were genetically modified to express the cytochrome P450 subenzyme 2B1 under the control of a cytomegalovirus (CMV) immediate early promoter. This CYP2B1 gene converts ifosfamide to its active cytotoxic compounds, phosphoramide mustard, which alkylates DNA, and acrolein, which alkylates proteins. The cells were then encapsulated in a cellulose sulphate formulation and implanted into preestablished tumors derived from a human pancreatic tumor cell line. Intraperitoneal administration of low-dose ifosfamide to tumor bearing mice that received the encapsulated cells results in partial or even complete tumor ablation. Such an in situ chemotherapy strategy utilizing genetically modified cells in an immunoprotected environment may prove useful for solid tumor therapy in man.

The prognosis of pancreatic adenocarcinoma is poor and current treatment ineffective. A novel treatment strategy is described here using a mouse model system for pancreatic cancer. Cells that have been genetically modified to express the cytochrome P450 2B1 enzyme are encapsulated in cellulose sulphate and implanted into pre-established tumours derived from human pancreatic cells. Cytochrome P450 2B1 converts the chemotherapeutic agent ifosfamide to toxic metabolites. Administration of ifosfamide to tumour-bearing mice that were recipients of implanted encapsulated cells results in partial or even complete tumour ablation. These results suggest that in situ chemotherapy with genetically modified cells in an immunoprotected environment may prove useful for application in man.

CD2 is a co-stimulatory receptor involved in T cell activation. Here we report on immunosuppressive effects of three mouse CD2 monoclonal antibodies (OX34, OX54, OX55) directed against non-overlapping epitopes of the rat CD2 receptor on various modes of T cell activation in vitro and in vivo. Although non-ligand-blocking OX54 and OX55, in concert, activated T cells through CD2 in vitro, they individually suppressed the mixed lymphocyte reaction (MLR) and significantly prolonged allograft survival after rat heart transplantation in vivo. Phenotype analysis revealed that OX55 significantly down-modulated CD2 in vivo, whereas OX54 depleted T cells. Graft rejection coincided with re-expression of CD2 and clearance of OX55 from serum, whereas T cell depletion by OX54 outlasted the period of graft survival. The most suppressive antibody, OX34, down-modulated CD2 and inhibited T cell activation through the TCR or CD2 and the MLR and prolonged median allograft survival time from 7 days in controls to 45 days in the absence of any additional treatment. Graft survival was clearly dose dependent and correlated with the duration of CD2 down-modulation and the presence of circulating CD2 antibody in serum. Importantly, the specific antibody production to a T cell-dependent antigen as demonstrated by immunization with keyhole limpet hemocyanin in vivo remained unaffected after treatment with OX34. These results demonstrate the pivotal role of CD2 signaling in mediating allogeneic immune reactions after vascularized organ transplantation while allowing specific humoral immune responses in vivo.

Epidemiological data show an association between Helicobacter pylori gastritis and gastric carcinoma. However, most people infected with H. pylori do not develop gastric cancer. We have therefore evaluated histological criteria indicating an increased risk for gastric cancer. H. pylori gastritis was investigated in 117 patients with small (∅

CD48, the murine homolog of human CD58, binds to CD2 in rats and mice. Whereas inhibition of CD2 signaling leads to profound immunosuppression, no information is available on CD48-targeted therapy in the rat. We could show that anti-CD2 treatment (OX34) efficiently inhibited TCR-driven as well as CD2-mediated proliferation, whereas blocking of ligand binding (OX45) remained completely uneffective. Inhibition of allogeneic MLR by OX45 turned out to be due to induction of unspecific suppressive mechanisms. In vivo, OX45 failed to prolong rat heart allograft survival in contrast to that seen with OX34. Grafts were rejected despite persistent and complete downmodulation of CD48 on lymphocytes without any cell depleting effect, rendering receptor/ligand interactions physically impossible. Combined application of CD2 and CD48 mAb did not enhance immunosuppression induced by CD2 mAb alone. Provided that there is no alternative CD2 ligand in the rat, we conclude that CD2-directed immunotherapy is mediated by suppressive events induced by modulation of the CD2 receptor (“negative signaling”) rather than by mere disruption of the CD2–CD48 interaction.

For the treatment of acute ischemic stroke (AIS), IV recominant tissue plasminogen activator (rtPA) has proven efficacy n eligible patients, but carries an increased risk of intracereral hemorrhage [1]. Preexisting anticoagulation, including the irect thrombin inhibitor dabigatran, usually contraindicates the se of rtPA. This notwithstanding, several single cases of IV tPA treatment in patients with AIS under dabigatran have een reported, with mixed results [2]. Endovascular therapy has merged as a potential, yet still experimental, alternative treatment ption for a number of clinical scenarios with contraindications o IV rtPA [3]. We report successful rescue thrombectomy in two patients who uffered AIS under treatment with dabigatran for atrial fibrillation AF).

BackgroundAdolescents with type 1 diabetes mellitus (T1DM) often show low adherence to complex insulin regimens, leading to poor glycemic control. The benefit of pharmaceutical care in adults with diabetes mellitus type 2 (T2DM) has been widely explored; however, evidence in adolescents with T1DM remains scarce.ObjectiveTo evaluate the impact of pharmaceutical care in adolescents with T1DM provided by a multidisciplinary team on multiple important clinical outcomes.SettingAt the outpatient Helios Paediatric Clinic and at the 12 regular community pharmacies of the study patients with 14 pharmacists in the Krefeld area, Germany, and at the University Pediatric Clinic with one pharmacist on-site in Sarajevo, Bosnia-Herzegovina.MethodsA randomized, controlled, prospective, multicenter study in 68 adolescents with T1DM. The intervention group received monthly structured pharmaceutical care delivered by pharmacists plus supplementary phone calls for 6 months. The control group received usual diabetic care. Data were collected at baseline and after 3 and 6 months. In addition, HbA1c was measured after 12 months.Main outcome measures The between-group difference in the change from baseline in glycosylated hemoglobin (HbA1c), the number of severe hypoglycemic events in both groups, and patient well-being in the intervention group.ResultsThe improvement from baseline in HbA1c was significantly greater in the intervention group than in the control group after 6 months (change from baseline −0.54 vs. +0.32%, p=0.0075), even after adjustment for country-specific variables (p=0.0078). However, the effect was more pronounced after only 3 months (−1.09 vs. +0.23%, p=0.00002). There was no significant between-group difference in the number of severe hypoglycemia events. After 6 months, the well-being according to the WHO-5 index in the intervention group increased significantly from 52.8% to 63.3%. After 12 months the mean total HbA1c remained significantly reduced in the intervention compared to the control group (8.6% vs. 9.5%, p=0.0184).ConclusionThe improved outcomes seen in this study provide new evidence that pharmaceutical care adds value to the management of T1DM in adolescents. However, the optimal methods of achieving sustained long-term improvements in this specific patient population require further study.

The acronym MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) belies the true scope of one of the most prevalent mitochondriopathies in adults. While the original description focused on neuromuscular symptoms, we now recognize this syndrome as genetically well defined but phenotypically profoundly heterogeneous, as exemplified by our experience. Here we report the case of a man who initially presented in 1986. In hindsight, his was a classic manifestation of MELAS, but the illness was ascribed to an ill-defined viral encephalitis. Over the years, diabetes and hearing impairment developed and his functional status deteriorated progressively. It took the quarter of a century to arrive at the correct diagnosis. It is worthwhile to keep an open mind when dealing with chronically ill patients with a seemingly clear-cut diagnosis.

Divalent cations like Mn2+ are known to strongly influence the integrin affinity to ligands and – in consequence – cell adhesion to extracellular matrix proteins. Therefore, divalent cation supplementation of biomaterials could be a promising approach to improve the ingrowth and the integration of implants. We were interested, whether manganese ions affect cellular functions like spreading, proliferation as well as gene expression in human osteoblasts. MG-63 osteoblastic cells were cultured in DMEM with 10% FCS. MnCl2 was added at a concentration range of 0.01–0.5 mM for 24 h and 48 h. Spreading (cell area in μm2) of PKH26-stained cells (cell membrane dye) was analyzed using confocal microscopy. Cell proliferation was measured by flow cytometry. Quantification of the phosphorylation status of signaling proteins was estimated using the Bio-Plex 200 system. Gene expression of osteogenic markers at the mRNA and protein level was analyzed by quantitative real time RT-PCR and Western blot, respectively. The results demonstrated that at higher concentrations of Mn2+ cells revealed a spindle shaped morphology. Further analyses indicated a reduced spreading, proliferation as well as phosphorylation of signaling proteins due to the influence of Mn2+ in a concentration-dependent manner. Although expression of bone sialo protein (BSP) at the mRNA level increased both after 24 h and 48 h in the presence of manganese, no increased expression of BSP was detected at the protein level. The expression of alkaline phosphatase (ALP) and collagen 1 (Col 1) mRNA decreased at >0.1 mM MnCl2. We speculate that the effect of manganese cations on cell functions is strongly concentration-dependent and the release of manganese when incorporated in a biomaterial surface has to be thoroughly adjusted.

To the Editor The development of an acute ischemic stroke (AIS) despite anticoagulant therapy has been called an ‘anticoagulation failure’ (1). In their case report, Tabata et al. (2) reviewed the literature pertaining to the use of intravenous thrombolysis in patients on the new oral anticoagulant (NOAC), dabigatran, presenting with AIS. Their article illustrates the challenges that clinicians face in trying to treat patients in situations where there are no evidence-based recommendations. Given the rapidly growing popularity of the direct oral anticoagulants, stroke neurologists are likely to be confronted with scenarios of this sort more frequently in the near future. All in all, the available case reports about thrombolysis in stroke patients on dabigatran paint a mixed picture, and indicate that there is still a significant risk of bleeding. In a recent review, Epple and Steiner expertly discussed the practical issues inherent to this therapeutic dilemma (1). The authors advised caution and concluded that “it is unlikely that patients on NOAC who experience an AIS would be suitable for thrombolysis, unless they have missed their NOAC dose for the last 24-48 hours”. For this reason, we would like to point to a different treatment option. We and others (3, 4) have shown that neurointerventional recanalization might constitute a promising treatment alternative in centers with high neuroradiological expertise. An endovascular approach using mechanical clot extraction devices does not expose patients to thrombolytic agents. Hence, a lower risk of intracranial hemorrhage is conceivable. All three cases reported so far showed a good clinical outcome. An asymptomatic intimal tear with transient extravasation of contrast medium was deemed likely in the first patient, and represents the only complication reported. As rightly commented by Hankey (5), such a “simple solution” remains unproven in terms of whether it is a safe and effective alternative to thrombolysis. Nevertheless, physicians occasionally face complex clinical situations and patient subgroups for which information derived from controlled clinical trials cannot be applied. We believe that case reports like those by Moey et al. and our group could form the basis of future observational studies. In our opinion, endovascular revascularization might emerge as a valuable treatment option for AIS patients on treatment with dabigatran, and possibly other novel oral anticoagulants.

Chronic Helicobacter pylori infection has been identified as a major risk factor for the subsequent development of gastric carcinoma On the basis of seroepidemiological studies the relative risk for infected persons was estimated to range between 3 and 6. Our study attempted to determine the relative risk of gastric carcinoma in H. pylori-infected individuals based on the histological evaluation of gastritis in gastric carcinoma patients in the light of a declining prevalence of H. pylori infection in Western countries. We histologically determined the H. pylori infection rate in 215 patients with early gastric carcinoma (tumor stage pT1), and compared it with that of 215 asymptomatic persons matched by age and sex who were tested by the 13C urea breath test. On the basis of these data an odds ratio of 16.7 (CI 9.6-29.1) was calculated for the relative risk of developing gastric carcinoma in H. pylori-infected people. The histological diagnosis of gastritis permits a separate risk assessment for patients with autoimmune gastritis, and by excluding these patients from the analysis we calculated an odds ratio for H. pylori-infected persons of 150 (CI 36.4-622.9). The endoscopic-histological diagnosis of H. pylori infection is associated with an increased risk of the subsequent development of gastric carcinoma of approximately 150-fold compared with H. pylori-negative patients who do not have chronic atrophic corpus gastritis of the autoimmune type (type A gastritis).

Background: The therapeutic efficacy of intratumoral instillation of genetically engineered, CYP2B1-expressing, microencapsulated cells in combination with ifosfamide had been previously demonstrated in xenografted human pancreatic ductal carcinomas [Gene Ther 1998;5:1070–1078]. Prior to a clinical study, the feasibility of an intra-arterial application of microencapsulated cells to the pancreas and its consequences to the organ had to be evaluated. Material and Methods: Microencapsulated, CYP2B1-producing cells were instilled both in vivo (transfemoral angiographical access) and in vitro (perfusion model) in the splenic lobe of the pig pancreas. In vivo, animals were monitored clinically for 7 days, then treated with ifosfamide and sacrificed. In vitro, ifosfamide was administered intra-arterially. Results: In all animals, 100 microcapsules could be instilled safely via the femoral route without clinical, biochemical or histological signs of pancreatitis. Histological examination revealed partial obstruction of small arteries by the capsules, without causing any parenchymal damage. In vitro, instillation reduced blood flow by half. Ifosfamide, also in combination with the capsules, did not add any damage to the pancreas. Conclusion: Intra-arterial instillation of microencapsulated cells to the pig pancreas is feasible and safe. Neither pancreatitis, foreign body reactions nor circulatory disturbances were observed. Clinical application of this genetically enhanced chemotherapeutic method seems possible.

Background: Mutations in p53 and ras genes are frequent in pancreatic carcinoma. Several ras mutations are consistently detected in the pancreatic juice from patients with chronic pancreatitis. The p53 gene mutations have been detected occasionally in chronic pancreatitis tissue. It was the aim of this study to evaluate the presence and clinical significance of p53 and ras mutations in clinical pancreatic juice samples from patients with chronic pancreatitis. Methods: Pancreatic juice was obtained from 66 patients with chronic pancreatitis and no evidence of pancreatic carcinoma (51 men, 15 women; age 17-86 years [mean 49.6 ± 12.9]). Patients were followed prospectively for 26 ± 3 (4-54) months. Detection of p53 gene mutations was by temperature gradient gel electrophoresis (TGGE) and single strand conformation polymorphism (SSCP) for exons 5-8. Analysis of ras mutations was performed by SSCP/polymerase chain reaction, restriction fragment length polymorphism/polymerase chain reaction. All mutations were confirmed by sequencing. Results: Five of 66 (7.5%) pancreatic juice samples contained p53 mutations, and ras mutations were detected in 6 cases (9%). Cytology was negative in all cases. No pancreatic carcinoma developed during follow-up and neither cancer cells nor preneoplastic lesions could be detected histologically in resected specimens. Although no correlation between p53 mutations and duration of pancreatitis or drinking habits was found, K- ras mutations correlated with both heavy smoking and severity of the disease. Conclusion: p53 and ras mutations can be detected in a minority of pancreatic juice samples from patients with chronic pancreatitis in the absence of malignancy. (Gastrointest Endosc 2001;53:734-43.)

Altered expression of cell surface molecules and the overexpression of growth factors and their receptors may be involved in malignant progression of human exocrine pancreatic cancer. CD44 is a transmembrane glycoprotein postulated to play a functional role in tumor cell metastasis. A “standard” form of CD44 (CD44st) binds hyaluronan (HA) and is reported to be overexpressed in pancreatic tumors.2 Alternative splicing gives rise to numerous CD44 isoforms. Variants containing the exon v6 appear to be of major importance for metastatic behavior in a rat pancreas carcinoma model.1 The published data about the expression of CD44st and CD44 isoforms in pancreatic cancer are contradictive.2–4 The regulation of CD44 expression is nearly unknown. Recently, a study showed a possible role of basic fibroblast growth factor (bFGF) in endothelial cells. It has been reported that bFGF and its high-affinity receptor (FGF-R) is abundantly present in pancreatic cancer.5 bFGF is one of the heparin-binding growth factors (FGF, epidermal growth factor (EGF)). The interaction with heparin protects bFGF from proteolytic degradation. Glycosaminoglycan heparin influences the binding of bFGF to the high-affinity receptor, too. Heparin and heparin derivatives can regulate tumor growth and inhibit metastatic behavior. The mechanisms of these regulation processes are poorly understood. Heparin is found exclusively in mast cells. Interestingly, mast cells accumulate by surrounding various neoplastic tissues. We asked whether bFGF alone, bFGF in combination with heparin, heparin alone or the CD44-ligand HA—a member of the glycosaminoglycans—modulates the expression of CD44. Therefore, we used the pancreatic adenocarcinoma cell lines PancTu and Panc-1 (both grade II–III). Furthermore, we investigated the effects of bFGF/heparin on proliferation behavior.

Although pancreatic adenocarcinoma has become one of the best characterized malignant diseases, severe diagnostic and therapeutic problems are still associated with this disease. The establishment of a molecular model of pancreatic carcinogenesis may provide tools that could result in earlier diagnosis of this disease and, in turn, improves prognosis. Since pancreatic adenocarcinoma seems to originate in epithelial cells in the pancreatic ducts, cultivation of native pancreatic duct epithelial cells (PDEC) is the initial step in the establishment of an in vitro model of pancreatic carcinogenesis. As these native cells survive only a short period in culture, the aim of this study was to establish a stable pancreatic duct cell line by immortalization with the SV40 large T antigen. Furthermore, initial steps in pancreatic carcinogenesis should possibly be imitated by additional transfections of mutated ki-ras and/or mutated p53 genes. By optimization of the isolation protocol and the culture medium, yield as well as proliferative activity of isolated PDEC was increased considerably. Transfection of SV40 large T antigen resulted in an increase in the proliferative lifetime of the isolated cells, but no real immortal phenotype was obtained. Moreover, one step in the transformation from the normal to the malignant phenotype was imitated successfully by additional transfection of mutated ki-ras.

Matthias Lohr (principal investigator) · Zoltan Tibor Bago · Helga Bergmeister · Manfred Ceijna · Mathias Freund · Wolfgang Gelbmann · Walter H. Gunzburg · Ralf Jesnowski · Johannes Hain · Karlheinz Hauenstein Wolfgang Henninger · Anne Hoffmeyer · Peter Karle · Jens-Christian Kroger · Gunther Kundt · Stefan Liebe Udo Losert · Petra Muller · Alexander Probst · Katrin Puschel · Matthias Renner · Renate Renz · Robert Saller Brian Salmons · Maximilian Schuh · Ilse Schwendenwein · Kerstin von Rombs · Thomas Wagner · Ingrid Walter (coinvestigators)

The prognosis of pancreatic adenocarcinoma is poor and current treatment is for the most part ineffective. We describe here a novel treatment strategy using a mouse model system for pancreatic cancer. Human embryonic epithelial cells have been genetically modified to express the cytochrome P450 2B1 enzyme under the control of a CMV immediate-early promoter. This CYP2B1 gene converts oxazaphosphorines (ifosfamide or cyclophosphamide) to their active cytotoxic compounds, phosphoramide mustard, which alkylates DNA, and acrolein, which alkylates proteins. A number of assays were performed to demonstrate the CYP2B1 gene function as well as toxic effects on neighbouring cells (bystander effect). The cells were then encapsulated in a cellulose sulphate formulation shown to be well tolerated in the pancreas of immunocompetent mice, and injected 1 cm away from pre-established tumours derived from a human pancreatic tumour cell line (PaCa-44). Intraperitoneal administration of low-dose ifosfamide to tumour bearing mice that received the encapsulated cells results in partial or even complete tumour ablation. Such an in situ chemotherapy strategy utilizing genetically modified cells in an immuno-protected environment may prove useful for solid tumour therapy in man.

To the Editor With interest we read the article by Rikimaru and co-workers reporting the effects of taurine, a potential new treatment for an important mitochondrial disorder (1). Two clinical cases were presented, thereby corroborating the laboratory findings. The medication regimens of these patients with the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) who harbored the typical 3243A>G mutation were switched from phenytoin to valproic acid (VPA) to treat ongoing seizure activity early in the course of the disease. In case 1, seizure control was not achieved until taurine supplementation was commenced the following year. Further stroke-like episodes ensued in case 2, before taurine was added. We would like to stress that theoretical concerns and anecdotal evidence caution against the use of VPA in mitochondrial disorders (2-4). In our view, not commenting on this matter would constitute a small, albeit relevant omission. VPA is known to interfere with intermediary metabolism, thereby affecting mitochondria structurally and functionally (3). In case reports, symptoms are paradoxically aggravated or triggered by VPA (3, 4). Hence, some renowned experts discourage its use in this setting (5). Admittedly, anticonvulsive treatment options were more limited in 1994 and 2001, respectively. In the meantime, however, evidence on the potentially deleterious effects of VPA in patients with mitochondriopathies has accumulated, while the antiepileptic armamentarium has broadened. Therefore, in a contemporary article on this topic, we feel a word of warning is in order.

The CD2 receptor is an important costimulatory molecule in T cell activation. Its ligand CD48 in rodents is supposed to be a homologue of human CD58, because of its similarities in structure and distribution. We evaluated the immunosuppressive activity of CD2/CD48-directed therapy in vitro and in vivo for the efficacy in prolonging rat heart allograft survival in a high responder transplant model. CD2-directed monoclonal antibody (mAb) therapy significantly prolonged median survival time to 45 days (P < 0.001). Suppression was mediated by down-modulation of CD2 below 20% on lymph node cells without considerable cell depletion. In contrast, CD48 mAb could not prolong graft survival. Rejection occurred in the presence of complete CD48 modulation and, therefore, despite disruption of the CD2–CD48 interaction. CD48 mAb failed to inhibit lymphocyte activation via a mitogenic pair of CD2 mAbs and inhibited mixed lymphocyte reaction (MLR) only by an unspecific mechanism. In conclusion, our results suggest a negative regulatory signal transduction by inhibitory CD2 mAbs and argue against a pivotal role of mere disruption of the CD2–CD48 interaction in CD2-mediated immunosuppression.

We have examined a series of 37 oropharyngeal squamous cell carcinomas for the presence of HPV 6/11, 16, and 18 DNA by polymerase chain reaction (PCR)/Southern blotting and for p53 alterations by immunohistochemistry and mutation screening with temperature gradient gel electrophoresis (TGGE). HPV sequences were found in a total of 26 of 37 cancers (70.3%), most frequently HPV 16 (20/37) followed by HPV 18 (11/37). Double infections with HPV 16 and 18 were present in 5 tumours. p53 accumulation was detectable immunohistochemically in 21 of 37 carcinomas (56.8%). There were remarkable differences in the distribution of immunoreactive tumour cells in relation to the tumour grade. A mutation screening for p53 by TGGE, directed to the amplified exons 5-8, revealed p53 mutations in 14 of 37 carcinomas (37.8%). Mutations in two different exons were present in 3 tumours, 11 tumours being hit once. Exon 7 was mutated in 6 carcinomas, exons 5 and 8 in 4 cases, and exon 6 in 3 cases. When grouping the tumours with p53 mutation according to their HPV state, HPV-positive cases showed slightly more mutations (11/26) than HPV-negative cases (3/11). Only 5 of 37 carcinomas (13.5%) contained neither HPV DNA nor p53 alterations. Our results indicate that high-risk HPV and p53 mutations frequently coexist in oropharyngeal carcinomas, in contrast to genital tumours, notably carcinomas of the cervix uteri. This may reflect different pathways in carcinogenesis in squamous cell epithelium from different sites.

Human papillomavirus (HPV) infections in oral carcinomas and normal oral mucosa were studied by consensus primer screening and typing for HPV types 6/11, 16 and 18 DNA. After polymerase chain reaction (PCR) the DNA species of interest were identified by Southern blot hybridization with digoxigenin-labeled oligonucleotide probes. Frozen tissue and scrapings were equally suitable for HPV testing and yielded high HPV detection rates in carcinomas. By comparison, HPV analysis of paraffin-embedded material was much less efficient. HPV were demonstrated in 61.5% (16/26) of oral squamous cell carcinomas, high risk HPV 16 and 18 being the preferential types. The frequency of HPV detection in non-neoplastic mucosa of tumor patients decreased clearly with increasing distance from the tumor (range 26.9-3.8%) suggesting focal HPV infections. In contrast, normal buccal mucosa of a group of healthy volunteers contained HPV DNA only in 1% (1/97).

Pancreatic cancer can seldom be resected, and chemotherapy has only a limited effect on survival or tumour load. We did a phase I/II trial in 14 patients with pancreatic cancer to assess the safety of local activation of low-dose ifosfamide. We encapsulated genetically modified allogeneic cells, which expressed a cytochrome P450 enzyme, in cellulose sulphate and delivered them by supraselective angiography to the tumour vasculature. These cells locally activated systemically administered ifosfamide. The tumours of four patients regressed after treatment, and those of the other ten individuals who completed the study remained stable. Median survival was doubled in the treatment group by comparison with historic controls, and 1-year survival rate was three times better. Further studies of this cell-therapy-based treatment combined with chemotherapy for inoperable pancreatic cancer are warranted.

Monosynaptic evoked field potentials (MEFP) were recorded in the dentate gyrus of male Wistar rats upon stimulation of either the perforant path or the commissural system. While the perforant path potential exhibited in acute experiments a clear reversal point of the field excitatory postsynaptic potential (EPSP) and population spike when protruding the registration electrode from the hippocampal fissura to the hilus of the dentate gyrus, the simultaneously registered commissural potential elicited by stimulation of the contralateral hilus showed no reversal of the negative monophasic wave but merely an amplitude maximum 40 microns above the reversal point of the perforant path potential. Frequency-related changes of the MEFPs during short tetanic stimulation with 15 Hz both in acute and chronic experiments, revealed differences in the properties of the input systems in that the commissural potential exhibited a clear frequency potentiation whereas the perforant path potential showed frequency depression. Pronounced long-term potentiation of the perforant path potential induced by 4 trains of tetanizing stimuli and lasting up to 72 h was accompanied by a long-term heterosynaptic depression of the commissural potential for up to 7 days after tetanization. Both the different frequency-related changes of the inputs and the extremely long duration of the heterosynaptic depression are discussed with respect to their proposed functions in the mechanisms of functional plasticity.