Transcapillary fluid balance consequences of missing initial lymphatics studied in a mouse model of primary lymphoedema

To investigate the phenotypic consequences of a deranged lymphangiogenesis in relation to tissue fluid accumulation and the possible role of inflammation in the pathogenesis of lymphoedema, we measured determinants of transcapillary fluid filtration and inflammatory mediators in the interstitial fluid in genetically engineered Chy mice, a model for primary congenital lymphoedema (Milroy's disease). Although initial lymphatics were not present in dermis in any of the areas studied (fore paw, hind paw, thigh and back skin) interstitial fluid pressure ( P _if), measured with micropipettes, and tissue fluid volumes were significantly increased only in the areas with visible swelling – the fore and hind paw, whereas interstitial colloid osmotic pressure (COP_if) was increased in all the skin areas examined. A volume load of 15% of body weight resulted in a more pronounced increase in P _if as well as a four-fold increase in interstitial fluid volume in Chy relative to wild-type (wt) mice, showing the quantitative importance of lymphatics for fluid homeostasis during acute perturbations. A similar level of proinflammatory markers in interstitial fluid in early established lymphoedema (3–4 months) in Chy and wt suggests that inflammation does not have a major pathogenetic role for the development of lymphoedema, whereas a reduced level of the immunomodulatory cytokine interleukin (IL)-4 may result in a reduced immunological defence ability and thus lead to the increase in inflammatory cytokines IL-2 and IL-6 observed at a later stage (11–13 months). Our data suggest that primary lymphoedema results in a high interstitial fluid protein concentration that does not induce an interstitial inflammatory reaction per se , and furthermore shows the paramount importance of the initial lymphatics in tissue fluid homeostasis, especially during perturbations of transcapillary fluid balance.     

Down-regulation of inducible nitric oxide synthase (iNOS) in rat with congenital hydronephrosis

BACKGROUND: Most of our knowledge concerning obstructive uropathy has been derived mainly from surgically manipulated animal models, and the pathogenesis of congenital obstructive hydronephrosis is not fully elucidated. Nitric oxide (NO) acts as an important biological modulator with diverse physiological functions, which can be either toxic or protective depending on the situation. NO is synthesized from l-arginine by nitric oxide synthase, and in the kidney iNOS is expressed spontaneously. The aim of our study is to investigate the expression of iNOS protein and its relationship with tubulointerstitial fibrosis and tubular cell apoptosis in congenital hydronephrosis. METHODS: We conducted histological studies on 18 kidneys of six-week-old-rats from an inbred colony of congenital hydronephrosis with reference to the histological grading of the affected kidney, tubulointerstitial fibrosis, renal tubular atrophy, and tubular cell apoptosis. Renal transforming growth factor-beta1 (TGF-beta1) level was determined by a sandwich ELISA assay and the expression of iNOS was analyzed by western blotting. RESULTS: Most of the hydronephrotic kidneys were markedly enlarged with dilatation of the collecting system, parenchymal thinning, tubular atrophy, interstitial infiltration and fibrosis. Renal TGF-beta1 level was higher in hydronephrotic kidneys than normal control kidneys (364.81 +/- 52.60 vs. 221.19 +/- 22.53 pg/mg protein, P < 0.05). Tubular apoptotic score in hydronephrotic kidneys was also significantly higher than in the normal control kidneys (1.97 +/- 0.42 vs. 0.14 +/- 0.02/HPF, P < 0.01). The expression of iNOS protein was lower in the affected kidneys compared with the normal control kidneys (8.79 +/- 0.78 vs. 14.00 +/- 0.83 arbitrary unit, P < 0.01). There was a negative correlation between iNOS expression and histological grading in congenital hydronephrosis. The iNOS expression also correlated negatively with renal interstitial fibrosis, TGF-beta1 level and tubular cell apoptosis. CONCLUSION: Our study confirmed the down-regulation of iNOS expression in affected kidneys from rats with congenital hydronephrosis, in which the cytoprotective effect of NO may be lost or weakened.     

Small cell lung cancer in Norway. Should more patients have been offered surgical therapy?

OBJECTIVE: The final outcome of patients with small cell lung cancer (SCLC) is poor with an overall 5-year survival rate of less than 10%. Therefore, the question of surgery in patients with a technically-operable solitary tumor has been raised. The purpose of this study was to identify the proportion of patients with operable SCLC and to assess the prognosis of different treatment strategies. For patients who were operated, we compared the resection specimens from patients with more than 5-year survival with those with shorter survival to see whether the specimens belonged to different subclasses of SCLC. METHODS: In Norway all clinical and pathologic departments submit reports on cancer patients to the Cancer Registry. The Registry also has a law-regulated authority to collect supplemental information regarding diagnosis, treatment and outcome for all cancer patients from the hospitals in charge. All reports on patients diagnosed as having SCLC in limited disease or unknown stage during the time interval 1993-1999 were reviewed. Patients with a T2-tumor, in whom a pneumonectomy would have to be performed, were classified as potentially operable. Five-year relative survival was calculated for patients diagnosed in 1993-1997. RESULTS: During the actual period 2442 individuals with SCLC were identified. The majority was treated with conventional chemotherapy or concurrent chemoradiotherapy while 38 underwent surgical therapy. Following reclassification of 697 patients reported to have limited disease or unknown stage 180 were judged to be in stage I. In addition to the 38 resected patients 14 were considered fit for surgery technically and medically while 97 were found to be potentially operable treatment modalities apart from surgery yielded a 5-year survival rate <7%. For stage I (N=96) the rate was 11.3% in conventionally treated patients compared to 44.9% for those who underwent surgical resection. By pathological review of surgical specimens a diagnosis of SCLC was confirmed in all patients treated by surgery in the groups with long and short survival. CONCLUSION: This investigation demonstrates that patients with SCLC having a peripherally located tumor should be referred to surgery, as long time survival is far better than for conventionally treated patients.     

A protease inhibitor attenuates gastric erosions and microcirculatory disturbance in the early period after thermal injury in rats

The effects of camostat mesilate, a synthetic serine protease inhibitor on gastric microcirculation and active oxygen species generated by leucocytes from the gastric and jugular veins in the early period after thermal injury were assessed. Male Wistar rats were anaesthetized and a 30% full skin-thickness dorsal burn was inflicted. Camostat mesilate (100 mg/kg) was dissolved in distilled water and administered orally to rats 40 min before thermal injury (the camostat group). The control animals (the vehicle group) were administered distilled water orally. Rolling leucocytes as well as Monastral blue B deposits in venules were observed using in vivo microscopy. Active oxygen species were measured by chemiluminescence. Camostat mesilate decreased the total length of gastric erosion, venular deposits of Monastral blue B, and rolling of leucocytes in venules, and relatively increased luminol-dependent chemiluminescence activity generated by zymosan-stimulated leucocytes 15 min after thermal injury. These results suggest that serine proteases are involved in the formation of gastric erosions and gastric microcirculatory disturbance in the early period after thermal injury.     

人骨髓间质干细胞体外扩增和向内皮细胞定向诱导分化的研究

目的：体外分离、扩增成人骨髓间质干细胞（MSCs）和向内皮细胞（ECs）定向诱导分化，开辟心血管组织工程种子细胞的新来源。 方法： 采用Percoll(1 073 g/L)从正常成人骨髓中分离出MSCs，纯化和扩增后流式细胞仪鉴定其纯度；用血管内皮生长因子(VEGF)诱导MSCs向ECs分化，Ⅷ因子(vWF)免疫组化和透射电镜(TEM)鉴定细胞性质。 结果： 5.0×105个MSCs在体外扩增15代后，获得8.0×1012个MSCs，扩增了约1.6×107倍；MSCs在加入VEGF诱导培养大约14-21 d，80%-90%的诱导细胞对Ⅷ因子相关抗原呈阳性反应；TEM可观察到胞浆内有Weible-palade小体，证实为ECs。 结论： 成人骨髓MSCs在体外具有定向诱导分化为ECs的潜能，这为心脏组织工程瓣体外构建, 尤其是在小儿先天性心脏病组织工程研究中种子细胞的来源提供了可能性。     

Structure and Function of Plasmid pColD157 of Enterohemorrhagic Escherichia coli O157 and Its Distribution among Strains from Patients with Diarrhea and Hemolytic-Uremic Syndrome

In this study, pColD157, a 6.7-kb colicinogenic plasmid of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain CL40cu, was characterized by restriction mapping and determination of its complete nucleotide sequence. The sequence consists of 6,675 bp and shows a high degree of similarity to the nucleotide sequence of colicinogenic plasmids pColD-CA23 and pColK. Seven potential genes were located on pColD157, three of which were closely related (>97.9%) to the colicin D structural gene and the corresponding immunity and lysis genes of plasmid pColD-CA23, and these were therefore designated cda, cdi, and cdl, respectively, using the reference extension -CL40 for differentiation. The adjacent 3′ region is related to the origin of replication of pColD-CA23. In contrast, the remaining part of the plasmid harbors a cluster of genes, closely related to the mobilization genes of pColK, which is followed by a 0.3-kb stretch homologous to the pColK resolution function. These determinants were designated mbdA, mbdB, mbdC, and mbdD and cdr, respectively. Southern blot analysis was performed with a probe specific for the cda gene of pColD157 and two groups of EHEC O157:H7 isolates from patients with diarrhea or hemolytic-uremic syndrome resident in Germany. Whereas 16 of 46 E. coli O157 strains isolated between 1987 and 1991 harbored plasmid pColD157, only 1 of 50 strains isolated during 1996 carried this plasmid. In addition, all strains harboring plasmid pColD157 were shown to have colicinogenic activity.     

Elektronenmikroskopische Untersuchungen am embryonalen Mycetom der Kleinzikade Euscelis plebejus Fall. (Homoptera,Cicadina)

Zusammenfassung Die a-Symbionten von Euscelis plebejus treten während der Embryonalentwicklung ihres Wirts in zwei morphologischen Typen auf, die als Infektionsstadium und als vegetative Form beschrieben werden. Die Infektionsstadien teilen sich durch septenartiges Einwachsen der Symbiontenmembranen. Infektionsstadien von Zikadensymbionten waren bisher nur während der Ovarialinfektion aus den Mycetomen weiblicher Imagines bekannt. Die a-Infektionsformen sind gekennzeichnet durch ihr elektronendichtes Cytoplasma, eine ungleichmäßige Verteilung der Ribosomen mit einer ribosomenfreien Randzone und durch Membrankörper. Das Cytoplasma der vegetativen Symbionten erscheint elektronendurch lässiger. Die Ribosomen liegen homogen in der Cytoplasmagrundsubstanz verteilt.Die a-Symbionten liegen vom Zeitpunkt der Eiablage bis zum Eindringen in die a₁-Mycetocyten als Infektionsstadien vor. Nach der Aufnahme in die a₁-Mycetocyten nehmen sie das Aussehen vegetativer Formen an. Im transitorischen Mycetom erscheinen erneut Infektionsstadien während der Übersiedelung in die zweikernigen a₂-Mycetocyten.Jeder Symbiont wird von zwei eigenen Membranen umgeben. Beim Eindringen von Ooplasma in den Symbiontenball wird jeder Symbiont von einer Wirtsmembran umhüllt, die er bis zur Infektion der Wirtszelle behält. In den Mycetocyten umgibt eine offensichtlich vom Wirtscytoplasma erneut gebildete Hüllmembran die vegetativen Symbionten. In den vegetativen a-Symbionten tritt häufig ein mikrofibrillärer kristallartiger Einschlußkörper auf. — Kernäquivalentstrukturen waren in keinem Symbiontenstadium cytologisch mit Sicherheit nachzuweisen.

---

Electron microscopic studies on the embryonic mycetome of the leafhopper Euscelis plebejus fall. (Homoptera, cicadina)

Summary During the embryonic development of their host the a-symbiotes of the leafhopper Euscelis plebejus appear in two different morphological types: infective form and vegetative form. The infective forms are able to multiply by ingrowth of their membranes. Until now infective forms of leafhopper symbiotes were only known from the ovarial infection by adult female mycetomes. The infective form of the a-symbiote is characterized by its electron dense cytoplasm, an asymmetric distribution of the ribosomes, a ribosome-free border, and by membraneous bodies. The cytoplasm of the vegetative form is less electron dense and the ribosomes are scattered throughout the cytoplasm.From the time of oviposition until they enter the a₁-mycetocytes, the a-symbiotes are found as the infective form. After entering the a₁-mycetocytes they take on the appearance of the vegetative form. In the transitory mycetome the infective forms appear again during the transition into the binucleate a₂-mycetocytes.Each symbiote is surrounded by two unit membranes of its own. As the ooplasm penetrates the symbiote mass, each symbiote is also surrounded by a host membrane, which remains until the infection of the host cells. In the mycetocytes the vegetative symbiotes have a new membrane around them, which is obviously developed from the cytoplasm of the host cell. In the vegetative a-symbiotes there is frequently a paracrystalline inclusion. — It was impossible to demonstrate nucleoid structure with certainty in any symbiote form.

---

Abkürzungen a a-Symbionten - a_i a-Infektionsstadien - a_v vegetative a-Symbionten - a-Mc a-Mycetocyt - a-Z a-Zelle - eK elektronendichter Körper - ER endoplasmat. Retikulum - M₁ innere Symbiontenmembran - M₂ äußere Symbiontenmembran - M₃ Hüllmembran - Mf Mikrofibrillen - Mi Mitochondrien - Mk Membrankörper - Mt Mikrotubuli - Nu Zellkern - Py Kernpyknosen - Ri Ribosomen - Sb Symbiontenball - Sm Symbiontenmembranen - t t-Symbionten - t_v vegetative t-Symbionten - t-Mc t-Mycetocyt - t-Z t-Zelle - Zm Zellmembran Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.Herrn Prof. Dr. K. Sander danke ich für stete Förderung meiner Arbeit und Frau A. Kaufmann für die Einführung in die elektronenmikroskopische Technik.