Adhesion of Escherichia coli under flow conditions reveals potential novel effects of FimH mutations.

FimH-mediated adhesion of Escherichia coli to bladder epithelium is a prerequisite for urinary tract infections. FimH is also essential for blood-borne bacterial dissemination, but the mechanisms are poorly understood. The purpose of this study was to assess the influence of different FimH mutations on bacterial adhesion using a novel adhesion assay, which models the physiological flow conditions bacteria are exposed to. We introduced 12 different point mutations in the mannose binding pocket of FimH in an E. coli strain expressing type 1 fimbriae only (MSC95-FimH). We compared the bacterial adhesion of each mutant across several commonly used adhesion assays, including agglutination of yeast, adhesion to mono- and tri-mannosylated substrates, and static adhesion to bladder epithelial and endothelial cells. We performed a comparison of these assays to a novel method that we developed to study bacterial adhesion to mammalian cells under flow conditions. We showed that E. coli MSC95-FimH adheres more efficiently to microvascular endothelium than to bladder epithelium, and that only endothelium supports adhesion at physiological shear stress. The results confirmed that mannose binding pocket mutations abrogated adhesion. We demonstrated that FimH residues E50 and T53 are crucial for adhesion under flow conditions. The coating of endothelial cells on biochips and modelling of physiological flow conditions enabled us to identify FimH residues crucial for adhesion. These results provide novel insights into screening methods to determine the effect of FimH mutants and potentially FimH antagonists.

Anti-cancer activity of targeted pro-apoptotic peptides.

We have designed short peptides composed of two functional domains, one a tumor blood vessel 'homing' motif and the other a programmed cell death-inducing sequence, and synthesized them by simple peptide chemistry. The 'homing' domain was designed to guide the peptide to targeted cells and allow its internalization. The pro-apoptotic domain was designed to be nontoxic outside cells, but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. Although our prototypes contain only 21 and 26 residues, they were selectively toxic to angiogenic endothelial cells and showed anti-cancer activity in mice. This approach may yield new therapeutic agents.

First-line daratumumab shows high efficacy and tolerability even in advanced AL amyloidosis: the real-world experience.

BACKGROUND: Daratumumab was the first monoclonal CD38 antibody with single-agent activity approved for the treatment of multiple myeloma. Moreover, daratumumab demonstrated high response rates in relapsed immunoglobulin light-chain (AL) amyloidosis. PATIENTS AND METHODS: In our single-center retrospective real-life case series, we analyzed the efficacy and safety of daratumumab as first-line treatment. Daratumumab was administered with low-dose dexamethasone alone or in combination with other multiple myeloma therapeutics RESULTS: Fourteen patients were eligible, including nine patients with cardiac stage IIIa or IIIb. Overall hematologic response rate was 100%, with 64.3% achieving complete response after a median of 16 cycles of treatment. Median time to hematologic response was 1.4 months. Organ response rates were 45.5% after a median of 4.0 months and 66.7% after a median of 10.0 months, for heart and kidney involvement, respectively. After a median follow-up of 20.5 months, two patients underwent successful autologous stem cell transplantation (ASCT), while another three patients were in preparation for ASCT. Three patients remained on daratumumab at the last follow-up. There were no unexpected toxicities and no grade III or IV adverse events, although more than half of our patients were in stage IIIa or IIIb. CONCLUSION: Daratumumab proved to be highly effective in newly diagnosed AL amyloidosis with excellent hematologic and organ response rates, a remarkable safety profile, and good tolerability even in patients with advanced stage of disease.

A novel class of autoantigens of anti-neutrophil cytoplasmic antibodies in necrotizing and crescentic glomerulonephritis: the lysosomal membrane glycoprotein h-lamp-2 in neutrophil granulocytes and a related membrane protein in glomerular endothelial cells.

Necrotizing and crescentic glomerulonephritis (NCGN) is frequently associated with circulating antineutrophil cytoplasmic autoantibodies (ANCA). It is established that ANCA are specific for soluble enzymes of granules of polymorphonuclear neutrophil granulocytes (PMN), such as myeloperoxidase (MPO) or protease 3 (PR3). The purpose of this study was to identify membrane proteins of PMNs, and/or glomerular cells, as additional autoantigenic ANCA targets. When membrane protein fractions were prepared from PMNs and isolated human glomeruli, and immunoblotted with ANCA sera of NCGN patients, two bands with apparent molecular masses of 170 and 80-110 kD (gp170/80-110) were labeled in PMNs, and a 130-kD glycoprotein (gp130) in glomeruli. Gp130 was purified, and monoclonal and rabbit antibodies (Abs) were produced which showed the same double specificity as the patient's ANCA. Using these probes, evidence was provided that gp170/80-110 is identical with human lysosomal-associated membrane protein 2 (h-lamp-2), because both proteins were immunologically cross-reactive and screening of a cDNA expression library from human promyelocytic leukemia cells with anti-gp130 Ab yielded a clone derived from h-lamp-2. Gp170/80-110 was localized primarily in granule membranes of resting PMNs, and was translocated to the cell surfaces by activation with FMLP. By contrast, gp130 was localized in the surface membranes of endothelial cells of human glomerular and renal interstitial capillaries, rather than in lysosomes, as found for h-lamp-2. Potential clinical relevance of autoantibodies to gp170/80-110 and gp130 was assessed in a preliminary trial, in which ANCA sera of patients (n = 16) with NCGN were probed with purified or recombinant antigens. Specific reactivity was detected in approximately 90% of cases with active phases of NCGN, and frequently also in combination with autoantibodies specific for PR3 or MPO. Collectively, these data provide evidence that h-lamp-2 in PMNs and a different, structurally related 130-kD membrane protein on the cell surface of renal microvascular endothelial cells are autoantigenic targets for ANCA in patients with active NCGN.

Blood Lactate And Lactate Clearance: Refined Biomarker And Prognostic Marker In Burn Resuscitation.

Adequate resuscitation of acute burn patients is important to ensure end organ perfusion and oxygenation. The ideal marker to the endpoint of burn resuscitation is still not established. We aimed to evaluate the role of blood lactate and lactate clearance in burn resuscitation and their association with mortality and sepsis in burn patients. The retrospective study included patients (18-50 years) with thermal and scald burns with total body surface area of 30% to 60% over a period of 9 months who had achieved target urine output of at least 0.5ml/kg/hr within 24 hours of resuscitation. Patients were divided based on their admission blood lactate levels (Group A < 2 mmol/L and Group B > 2 mmol/L). Group B was further subdivided into Group B1 in whom blood lactate levels reached less than 2 mmol/L within 24 hours of burn resuscitation and Group B2 in whom it did not. Total patients included were 203. Mortality (M) and sepsis (S) rates in subgroup B2 were higher (M=57.9%; S=43.5%) and rates in subgroup B1 (M=25.8%; S=27.4%) were comparable to Group A (M=27.8%; S=26.4%). Persistent lactic acidosis at 24 hours was independently associated with significantly increased mortality and sepsis. Our data suggests a correlation of blood lactate levels and lactate clearance within 24 hours of admission with mortality and sepsis related to burn injury.

Un remplissage vasculaire adapté est nécessaire afin de préserver la perfusion et l'oxygénation tissulaires des brûlés. Le marqueur idéal de sa qualité reste à trouver. Nous avons évalué la lactatémie et l'élimination des lactates dans ce but, ainsi que leur corrélation avec la mortalité et le sepsis. Nous avons étudié rétrospectivement, sur 9 mois, 203 patients de 18 à 50 ans, brûlés sur 30 à 60% de SCT, ayant eu une diurèse horaire de plus de 0,5 mL/kg/h dans les 24 premières heures suivant leur brûlure. Le groupe A avait moins de 2 mmol/L de lactate à l'admission, le groupe B plus. Ce dernier groupe a été subdivisé en B1 (lactate redescendant à moins de 2 mmol/L dans les 24 premières heures) et B2 ne le faisant pas. La mortalité de B2 était plus élevée (57,8%) que A (27,8%) et B1 (25,8%), ces 2 derniers groupes étant comparables. De même, un sepsis survenait chez 43,5% des patients de B2 contre 27,4% pour B1 et 26,4% pour A. Plus que leur valeur initiale, c'est l'absence de décroissance dans les 24 premières heures des lactates qui est un marqueur de mauvais pronostic chez le brûlé.

Interdigitating dendritic cell sarcoma of the parotid gland.

Interdigitating dendritic cell sarcomas (IDCSs) are extremely uncommon tumours that arise predominantly in lymphoid tissue. We report a case of an IDCS arising in the parotid gland of a 73-year-old man. Clinically, a primary salivary gland tumour was suspected but fine needle aspiration cytology suggested a soft tissue tumour. A diagnosis of IDCS was made on histopathological examination of the resection specimen, with subsequent confirmation by electron microscopy. Given the extreme rarity of this tumour at this site, it is unlikely to be a common diagnostic problem, but the importance of multiple diagnostic modalities is emphasized. The findings of cytology, histology, immunohistochemistry and electron microscopy have not previously been described together in a single case report of this tumour.

Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis.

Phage that display a surface peptide with the NGR sequence motif home selectively to tumor vasculature in vivo. A drug coupled to an NGR peptide has more potent antitumor effects than the free drug [W. Arap et al., Science (Washington DC), 279: 377-380, 1998]. We show here that the receptor for the NGR peptides in tumor vasculature is aminopeptidase N (APN; also called CD13). NGR phage specifically bound to immunocaptured APN and to cells engineered to express APN on their surface. Antibodies against APN inhibited in vivo tumor homing by the NGR phage. Immunohistochemical staining showed that APN expression is up-regulated in endothelial cells within mouse and human tumors. In another tissue that undergoes angiogenesis, corpus luteum, blood vessels also expressed APN, but APN was not detected in blood vessels of various other normal tissues stained under the same conditions. APN antagonists specifically inhibited angiogenesis in chorioallantoic membranes and in the retina and suppressed tumor growth. Thus, APN is involved in angiogenesis and can serve as a target for delivering drugs into tumors and for inhibiting angiogenesis.

The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization.

Eph receptor tyrosine kinases and their ephrin ligands have been implicated in embryonic vascular development and in in vivo models of angiogenesis. Eph proteins may also regulate tumor neovascularization, but this role has not been previously investigated. To screen for Eph proteins expressed in tumor blood vessels, we used tumor xenografts grown in nude mice from MDA-MB-435 human breast cancer cells or KS1767 human Kaposi's sarcoma cells. By immunohistochemistry, the ephrin-A1 ligand and one of its receptors, EphA2, were detected throughout tumor vasculature. Double-labeling with anti-CD34 antibodies demonstrated that both ephrin-A1 and EphA2 were expressed in xenograft endothelial cells and also tumor cells. Furthermore, EphA2 was tyrosine-phosphorylated in the xenograft tumors, indicating that it was activated, presumably by interacting with ephrin-A1. Ephrin-A1 and EphA2 were also detected in both the vasculature and tumor cells of surgically removed human cancers. In an in vitro angiogenesis model, a dominant negative form of EphA2 inhibited capillary tube-like formation by human umbilical vein endothelial cells (HUVECs), demonstrating a requirement for EphA receptor signaling. These data suggest that ephrin-A1 and EphA2 play a role in human cancers, at least in part by influencing tumor neovascularization. Eph proteins may represent promising new targets for antiangiogenic cancer treatments.

Capillary haemangioma of the testis.

A case of testicular capillary haemangioma is reported and the importance of intraoperative examination of this very rare lesion emphasised. Capillary haemangioma of the testis can be similar to malignant testicular tumours on clinical presentation, as well as on ultrasonography and magnetic resonance imaging, and therefore should be included in the intraoperative differential diagnosis. Because of the benign nature of this lesion, conservative surgical treatment by means of tumour enucleation with preservation of the testis is possible, if intraoperative examination of frozen sections of representative tissue can be performed.

[Vascular kidney transplant rejection--is a duplex sonographic diagnosis possible?]. / Vaskuläre Nierentransplantatabstossung--Ist eine duplexsonographische Diagnose möglich?

The diagnostic value of quantitative Duplex Doppler sonography (DS) in renal allograft evaluation is being viewed increasingly critically. We undertook a retrospective analysis of DS in 51 consecutive patients to assess the capability of DS in the diagnosis of vascular rejection. At the time of renal allograft biopsy, a mean resistive index (RI) was calculated from Doppler measurements within main, segmental, interlobar and arcuate arteries and correlated with histological diagnosis. Our results indicate a low specificity (36% for RI greater than 0.7), low sensitivity (35% for RI greater than 0.9) as well as a low positive predictive value (54% for RI greater than 0.7) for the diagnosis of vascular rejection. Therefore, elevation of the RI is an unspecific finding in different causes of allograft dysfunction. Thus, renal biopsy to establish specific histologic diagnosis of allograft dysfunction remains mandatory.

[Ultrasound-guided fine-needle biopsy with an automatic full-incision system: the initial experiences and comparison with a conventional biopsy gun]. / Ultraschallgezielte Feinnadelbiopsie mit einem automatischen Vollschnittsystem: erste Erfahrungen und Vergleich mit einer konventionellen Biopsiepistole.

The purpose of this study was to evaluate the efficacy of a new automatic biopsy device (Autovac, Angiomed, Karlsruhe, Germany) in ultrasound (US) guided biopsies of focal abdominal lesions. 50 consecutive patients with focal abdominal lesions underwent US guided biopsies. In the first 24 patients, needle passes were performed with the Autovac system (outer diameter 0.95 mm) as well as with the Biopty gun (outer diameter 0.9 mm) (Bard Covington, USA). The size and the quality of the histologic and the cytologic material obtained by both systems were evaluated by histopathologists blinded to the system used. Autovac yielded significantly more material (defined as the area of the obtained tissue cores) and a significantly higher quality score than did the Biopty system. 96% of the histologic specimen and 100% of the cytologic smears obtained with Autovac were diagnostic, compared to 70 and 81% with Biopty, respectively. With the exception of a short-time elevation of the blood pressure in one patient, no complications occurred. The results indicate an advantage of the automatic full-cut type system Autovac over the tru-cut type Biopty gun in US-guided biopsies of focal abdominal lesions.

[Ovarian tissue banking--primary results]. / 'Ovarian Tissue Banking' - erste Ergebnisse.

Lifelong hormone replacement therapy and infertility often follow oncological therapy in young women due to a dramatic reduction of the primordial follicle reserve. Reimplantation and function of frozen ovarian tissue slices were successfully demonstrated in animal models, but the durability is limited. Cryopreservation of a whole ovary could reestablish an almost normal ovarian function. Experiments with porcine ovaries showed histological viability. Cryopreservation and retransplantation of sheep ovaries should demonstrate success by hormonal response and pregnancy.

[Microscopic polyangiitis with eosinophilia--an overlap syndrome or separate disease entity? A case report and review of the literature]. / Mikroskopische Polyangiitis mit Eosinophilie--Uberlappungssyndrom oder eigenständiges Krankheitsbild? Ein Fallbericht und Literaturübersicht.

Systemic vasculitides are potentially life-threatening diseases. Early and appropriate diagnosis based on case history, clinico-pathological features, and laboratory parameters, such as the presence of anti-neutrophil cytoplasmic antibodies (ANCA), is crucial for starting appropriate and, often, life-saving therapeutic measures. We report a 50-year-old female patient who presented with fever, arthralgias and hemoptysis. Skin signs included disseminated hemorrhagic pustules, ulcerations of oral and genital mucosa, subcutaneous nodules on arms and legs, and a pyoderma gangrenosum-like lesion on the right leg. Laboratory investigations revealed a peripheral eosinophilia and a positive cANCA titer. Histopathologic analysis of various biopsy specimens showed a granulomatous vasculitis in the subcutis, a nongranulomatous vasculitis with massive eosinophil infiltration in the lungs, and a segmental, necrotizing glomerulonephritis in the kidneys. Differential diagnosis included Wegener's granulomatosis, microscopic polyangiitis (MPA) and Churg-Strauss syndrome. MPA was diagnosed based on clinical and histopathological criteria. An interesting feature of this case was marked peripheral and tissue eosinophilia. Therapy consisted of cyclophosphamide and methylprednisolone. The patient went into a long-lasting clinical remission one month after starting therapy.

Molecular cloning and expression of a novel human trans-Golgi network glycoprotein, TGN51, that contains multiple tyrosine-containing motifs.

Previously, it has been shown that glycoproteins with approximately 130-kDa molecular mass react with antisera from patients with renal vasculitis (Kain, R., Matsui, K., Exner, M., Binder, S., Schaffner, G., Sommer, E. M., and Kerjaschki, D. (1995) J. Exp. Med. 181, 585-597). To search for a molecule that reacts with the antibodies, we screened a lambdagt11 human placental cDNA library. Two of the isolated clones were found to encode a putative counterpart of the rodent trans-Golgi network (TGN) glycoprotein 38, hTGN46, which has the tyrosine containing motif YQRL shared by mouse and rat TGN38. Moreover, reverse transcription-polymerase chain reaction analysis of hTGN46 transcripts and genomic analysis of a cDNA deposited as an expressed sequence tag in dbEST Data Base revealed that additional cDNAs exist that are produced by alternate usage of 3'-splice sites of intron III. Alternative splicing results in frame shifts and leads to novel larger translation products with one (for hTGN48) or two (for hTGN51) additional tyrosine-containing motifs. hTGN51 expressed in Chinese hamster ovary cells were localized to the trans-Golgi network, overlapping with beta-1,4-galactosyltransferase even after mutating the tyrosine-containing motif common to hTGN46. In contrast, mutated hTGN48 and hTGN46 are no longer retrieved to the TGN. These results strongly suggest that hTGN51 may have a unique function compared with hTGN46 or hTGN48 in shuttling between the cell surface and the TGN.

Immunological risk factors are solely responsible for primary non-function of renal allografts.

Primary non-function (PNF) of renal allografts has been attributed to various risk factors, among them immunological ones, as well as unfavourable preservation conditions. To investigate the impact of these risk factory on the occurrence of PNF, 1335 consecutive kidney transplants performed at a single centre over a 10-year period were analysed. All patients received immunosuppression based on cyclosporine. As the method of analysis a conditional stepwise logistic regression model was chosen, comparing each graft suffering PNF with its partner kidney retrieved from the same donor. Thus, all donor-related variables could be omitted from the analysis, as they are the same in every pair of grafts. Risk factors analysed included panel-reactive antibodies, number of pretransplant transfusions, pregnancies, number of prior transplants, cold and second warm ischaemia time, mismatches on HLA loci A, B and DR and recipient age. The overall incidence of PNF was 87 grafts (6.5%). One patient suffered immediate rejection due to transplantation of an ABO incompatible graft. This case was excluded from further analysis. PNF occurred three times in recipients of living related grafts, twice in recipients of en-bloc grafts and four times in grafts, in which the paired kidney was either not transplanted or shipped outside the Eurotransplant region, so that no paired graft was available for matched case-control analysis. Of the remaining 77 pairs, twice both organs of one donor failed immediately. The remaining 73 complete pairs were analysed. Two of the investigated risk factors have independently a significant impact on the occurrence of PNF. Increasing the number of pretransplant transfusions raises the relative risk of graft failure up to six fold (P=0.02), while a history of prior transplants bears a relative risk of 0.21E05 (P=0.005). Ischaemia has no significant impact on the occurrence of PNF. Our data strongly suggest that immunological rather than donor risk factors are responsible for the non-function of kidney grafts.

Altered Golgi localization of core 2 beta-1,6-N-acetylglucosaminyltransferase leads to decreased synthesis of branched O-glycans.

Mucin type O-glycans with core 2 branches are distinct from nonbranched O-glycans, and the amount of core 2 branched O-glycans changes dramatically during T cell differentiation. This oligosaccharide is synthesized only when core 2 beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) is present, and the expression of this glycosyltransferase is highly regulated. To understand how O-glycan synthesis is regulated by the orderly appearance of glycosyltransferases that form core 2 branched O-glycans, the subcellular localization of C2GnT was determined by using antibodies generated that are specific to C2GnT. The studies using confocal light microscopy demonstrated that C2GnT was localized mainly in cis to medial-cisternae of the Golgi. We then converted C2GnT to a trans-Golgi enzyme by replacing its Golgi retention signal with that of alpha-2,6-sialyltransferase, which resides in trans-Golgi. Chinese hamster ovary cells expressing wild type C2GnT and the chimeric C2GnT were then subjected to oligosaccharide analysis. The results obtained clearly indicate that the conversion of C2GnT into a trans-Golgi enzyme resulted in a substantial decrease of core 2 branched oligosaccharides. These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than alpha-2,3-sialyltransferase that competes with C2GnT for the common substrate. Furthermore, alteration of Golgi localization renders the chimeric C2GnT much less efficient in synthesizing core 2 branched oligosaccharides, indicating the critical role of orderly subcellular localization of glycosyltransferases.

Polyetiology of renal allograft dysfunction. Does calculation of the resistive index still make sense?

In 101 consecutive patients with renal allograft dysfunction a correlation of Duplex Doppler sonography (DDS) with histopathologic reports of simultaneously performed biopsies was made. Renal vascular impedance was estimated by calculating the resistive index (RI). A total of 290 different specific histologic diagnoses (mean 2.1 +/- 0.84 diagnoses/biopsy) was noted. With increasing time interval to transplantation, single diagnoses as cause of allograft dysfunction decreased. DDS could not reliably differentiate, exclude, or grade any of the common causes of renal allograft dysfunction like vascular and/or cellular rejection, chronic rejection, acute tubular necrosis, cyclosporin nephrotoxicity, relapse of glomerulonephritis and infection. Follow-up studies after established histologic diagnosis in 19 patients with persisting allograft dysfunction demonstrated a lack of sensitivity of DDS to significant superimposed causes of transplant malfunction. We conclude that biopsy is still necessary to direct proper therapy of renal allograft dysfunction.