Integrin expression on colorectal tumor cells growing as monolayers,as multicellular tumor spheroids,or in nude mice

In this study we compared the expression of integrin alpha chains 2, 3, 4, 5, 6, v and the beta chains 1, 3, 4 in 2 colorectal carcinoma cell lines (HRT-18 and CX-2), growing in confluent and subconfluent monolayer cultures, as multicellular tumor spheroids and in nude mice, using the immunofluorescence technique (confocal microscopy) and flow cytometry. The fast-growing cell line HRT-18 expressed, in confluent and subconfluent monolayer cultures, alpha 2, 3 and beta 1 with a continuous membranous staining pattern, whereas alpha v, alpha 6, and beta 4 were expressed continuously membranous in the intermediate and apical part of the cell layer, and clustered at focal contacts at the base of the cells. In spheroids and tumors of nude mice the focal pattern of alpha v, 6 and beta 4 was changed into a diffuse one. Using flow cytometry, the expression of alpha 3 was found to be reduced in spheroids of HRT-18. The slowly-growing cell line CX-2 expressed, under the same conditions in monolayer culture, alpha 6, beta 1 and beta 4, and very weakly alpha 2, 3, 5 and v. Alpha 3 was expressed in spheroids of CX-2 only at the outer rim where the cells proliferate. In contrast, alpha 2 and 5 were expressed mainly in the quiescent, non-proliferating area. Alpha 6 was reduced in spheroids of CX-2. In the nude mouse tumor of CX-2, alpha 5 was expressed only focally and very weakly, alpha 2 was no longer detectable, but alpha v appeared to be enhanced in a focal pattern. These data indicate that integrin expression of tumor cells depends upon the culture system and that integrin expression in multicellular tumor spheroids is more similar to the in vivo situation in nude mouse tumors. © 1995 Wiley-Liss, Inc.     

Monoclonal antibody enzyme-linked immunosorbent assay for identification of K99-positive Escherichia coli isolates from calves.

For Escherichia coli to produce diarrhea in animals it must possess the ability to attach to the epithelial cells of the intestine and to produce enterotoxins. Tests developed to differentiate pathogenic from nonpathogenic E. coli have relied on detection of adherence structures called pili or detection of the toxins. We utilized a monoclonal antibody to K99 pili in an enzyme-linked immunosorbent assay to detect the presence of K99 pili in E. coli isolated from calves. Twenty-three E. coli isolates that were known to be stable toxin positive were all shown to produce K99 pili. A 100% correlation also was shown between the presence of K99 antigen and production of stable toxin by E. coli isolates. Of the 251 isolates, 245 were negative by K99 enzyme-linked immunosorbent assay and stable toxin assay. The other six were positive on both tests. The enzyme-linked immunosorbent assay also was shown to be specific for K99 pili by antibody-blocking assays. The number of E. coli necessary for detecting K99 pili by enzyme-linked immunosorbent assay was determined to be 3.5 X 10(5) bacteria per ml.     

Prevalence and polymorphism of genes encoding translocated effector proteins among clinical isolates of Salmonella enterica

Pathogenic Salmonella enterica strains are capable of causing local and/or systemic infections. They employ two type III secretion systems to translocate an array of virulence-associated proteins (effector proteins) directly into the cytosol of target cells of the host. Earlier data had shown that changes in the repertoire of translocated effector proteins may contribute to the adaptation of Salmonella strains to new hosts and to the emergence of epidemic strains. Using PCR and Southern blot techniques the presence of and the polymorphism among the genes encoding the translocated effector proteins SopB, SopD, SopE, SopE2, SipA, SipB, SipC, AvrA, and SptP was studied in 71 phylogenetically well characterised S. enterica subspecies I (subspecies enterica) strains of the SARB collection and in 209 clinical and epidemic isolates of S. enterica subspecies I belonging to various serovars, phage types, and genotypes. All these Salmonella strains harbour all these respective genes with the exception of sopE and avrA which have been identified in only some of them. Several of the studied genes display genetic polymorphisms (RFLP). These RFLP patterns did not show a strict correlation with the genetic distance, the grouping genes in order to understand their role in the evolution of Salmonella as a pathogen.     

Expression of heat shock proteins 72/73 in human peritoneal mesothelial cells in vivo and in vitro

Leukocyte accumulation during peritonitis leads to an injurious microenvironment which is involved in the host defense reaction but is also thought to cause peritoneal damage. We tested the hypothesis that mesothelial cells (MC) respond to the injurious microenvironment during peritonitis by an increased expression of heat shock proteins (HSP 72/73), a basic way by which cells are protected against injury. Comparison of resting MC and activated MC during peritonitis in vivo by means of immunohistochemistry revealed an increased expression of HSP 72/73. As assessed by Western immunoblotting, incubation of MC in vitro with tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) caused a time-dependent induction of HSP 72/73 expression, which was maximal 6 h after stimulation. We suggest that the increased HSP 72/73 expression of MC during peritonitis is in part induced by TNF-alpha and IL-1beta and may exert a cell-protective function, lessening MC damage during peritonitis.     

Expression and function of beta(1) and beta(3) integrins of human mesothelial cells in vitro.

Mesothelial cells (MC) and extracellular matrix (ECM) components are thought to play a pivotal regulatory role during the inflammatory-reparative response of serosal membranes. Integrins are known to serve as cellular ECM receptors, but mesothelial integrin expression and its function, particularly its role for attachment to different ECM components, remain to be elucidated. The aim of the present study was to characterize the integrin expression of human omentum majus derived MC (HOMC) in vitro by immunohistochemistry and to investigate their functional significance with regard to HOMC adhesion to fibronectin (fn), vitronectin (vn), collagen IV (coll IV), and laminin (ln). Mesothelial cells in vitro strongly expressed beta(1), beta(3), alpha(2), alpha(3), alpha(5), and alpha(v) chains. A weak reactivity was found for alpha(1) and alpha(6), but no alpha(4) reactivity was detectable. Compared to the control, fn, vn, coll IV, and ln caused a significant 2.6-, 2.2-, 2-, and 1.6-fold increase of HOMC adhesion, respectively. Inhibition studies revealed that HOMC attachment to fn is mediated by alpha(5)beta(1), alpha(v)beta(1), and alpha(v)beta(3), with a synergistic effect of alpha(5)beta(1) and alpha(v)beta(3). Adhesion to vn is mediated by alpha(v)beta(1) and alpha(v)beta(3). Integrins alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) mediate adhesion to coll IV and ln. We suggest that the integrin expression and function of mesothelial cells described here play an important role in the interaction of MC with the ECM, particularly during the acute and chronic inflammatory-reparative response of serosal membranes.     

Consensus polymerase chain reaction and enzyme-linked immunosorbent assay for human papillomavirus detection and typing in cervical specimens.

Human papillomavirus (HPV) infection is common in cervical intraepithelial neoplasia (CIN). This study investigates HPV detection and typing assay based on polymerase chain reaction amplification of L1 open reading frame with general primers GP5/GP6, followed by enzyme-linked immunosorbent assay detection with type-specific DNA probes. To determine the sensitivity of this assay, formalin-fixed CaSki cells were used as reference cell lines. Fifty copies of viral DNA diluted in DNA from 100,000 noninfected cells could be detected. This assay was also investigated for HPV detection and typing of 67 cervical specimens diagnosed with with CIN III or carcinoma in situ (CIS) and their adjacent squamous epithelium. The CIN III lesions were infected in approximately 80% of the samples, 81% in the neighboring CIN II, and 68% in CIN I. The HPV infection was even detectable in 54% of nondysplastic epithelium located near a CIN III lesion.     

Hereditäre sensorische und autonome Neuropathie Typ IV (HSAN IV) als Ursache rezidivierender Fieberschübe

Zusammenfassung Hintergrund: Die heredit?re sensorische und autonome Neuropathie vom Typ IV (HSAN IV) ist eine seltene, autosomal-rezessiv vererbte Krankheit. Sie ist gekennzeichnet durch fehlendes Schmerzempfinden, rezidivierende Fieberschübe, Unf?higkeit zu schwitzen, mentale Retardierung mit muskul?rer Hypotonie, rezidivierende fleckige Erytheme und fehlende Hautr?tung nach lokaler Histaminapplikation. Fallbericht: Wir berichten über Zwillinge, die wegen hohen Fiebers vorgestellt wurden und bei denen wir diese Erkrankung diagnostizierten. Diskussion: Bei Patienten mit rezidivierendem Fieber unklarer Ursache genügt ein kurzer Test des Schmerzempfindens, um diese Neuropathie nicht zu übersehen.      

Renal haemodynamic changes, renal tubular function, sodium and water homeostatic hormones in patients with chronic glomerulonephritis and in healthy humans after intravenous infusion of amino acids

To determine whether renal reserve capacity was preserved inpatients with chronic glomerulonephritis with well-preservedkidney function, and how sodium was handled in proximal anddistal tubules, 13 healthy control subjects and 13 patientswith biopsy-verified chronic glomerulonephritis were studiedbefore and during a continous 120-min amino-acid infusion. Glomerularfiltration rate (GFR), renal plasma flow (RPF), and tubularfunction evaluated by the lithium clearance method, were determinedduring six clear ance periods of 30 min each. Plasma concentrationsof angiotensin II, atrial natriuretic peptide (ANP), aldosterone,arginine vasopressin (AVP), glucagon, amino acid and serum osmolalitywere determined before, 60, and 120 min after infusion. GFRand RPF increased about 10% in both groups; filtration fraction(FF) was unchanged. Proximal tubular reabsorption of sodiumand water decreased, and distal tubular reabsorption of sodiumand water increased, and thus the net excretion of sodium andwater was unchanged. Angiotensin II and aldosterone were reducedin control subjects, but not in the patients. ANP and glucagonincreased equally in both groups. Most amino acids increasedtwo- or threefold. It is concluded that renal reserve capacityand glomerulotubular balance are intact in patients with chronicglomerulonephritis with well-preserved renal function, but thereis an abnormal lack of suppression of the renin-angiotensin-aldosteronesystem in response to an amino acid infusion in these patients.