Standard lymph node dissection for bladder cancer: significant variability in the number of reported lymph nodes.

PURPOSE: We compared the nodal yield after histopathological examination of extended bilateral pelvic lymph node dissection specimens for bladder cancer at 2 hospitals. Surgery at each hospital was done by the same 4 staff urologists using a standardized extended bilateral pelvic lymph node dissection template. MATERIALS AND METHODS: All consecutive patients with bladder cancer who underwent extended bilateral pelvic lymph node dissection from January 1, 2007 to December 31, 2009 were included in this study. Specimens were sent for pathological evaluation in a minimum of 2 packages per side. At the 2 pathology departments specimens were processed according to institutional protocols. RESULTS: A total of 174 patients with a mean age of 62.7 years were included in analysis. At hospital 1 a mean of 16 lymph nodes were found after dissection vs a mean of 28 reported at hospital 2 (p <0.001). No significant differences were found in the number of tumor positive lymph nodes (p = 0.65). Mean lymph node density at hospitals 1 and 2 was 9.3% and 3.9%, respectively (p = 0.056). CONCLUSIONS: Despite equal anatomical clearance by the same experienced surgeons we report a statistically significant difference between 2 pathology departments where the number of lymph nodes was evaluated after extended bilateral pelvic lymph node dissection for bladder cancer. Unless standardized methods are agreed on by pathologists, the number of reported lymph nodes as an indicator of surgical quality and lymph node density as a prognostic factor should be used cautiously.

Bone formation and calcification by isolated osteoblastlike cells.

Two cell populations were isolated from calvaria of chick embryos: PF cells were liberated by collagenase treatment from the periosteum, OB cells from the periosteum-free calvarium. Both populations were cultured in plastic culture dishes. After 6 d of culture, monolayers of each cell type either were scraped off the culture dishes, transplanted on the chorio-allantoic membrane of 7-d-old quail eggs, and cultured there for 6 d, or were used for biochemical experiments. OB transplants proved capable of producing calcified bone matrix, whereas PF transplants formed only fibrous tissue. Biochemically, OB cells showed high cAMP production in the presence of parathyroid hormone (PTH), whereas cAMP production was not stimulated in PF cultures. Lactate production was stimulated by PTH in both populations although somewhat differently. Citrate decarboxylation was high in OB cells and was inhibited by PTH but was low in PF cells, where it was stimulated by the same hormone. The differences in hormonal response between the two cell types made it possible to conclude that PF cultures are relatively free of OB cells. The PF contamination in OB cultures was more difficult to assess. The experiments described in this report show that the OB population contains osteoblasts or osteoblastlike cells which are, under favorable circumstances, capable of bone formation.

Multinational development and validation of an early prediction model for delirium in ICU patients.

RATIONALE: Delirium incidence in intensive care unit (ICU) patients is high and associated with poor outcome. Identification of high-risk patients may facilitate its prevention. PURPOSE: To develop and validate a model based on data available at ICU admission to predict delirium development during a patient's complete ICU stay and to determine the predictive value of this model in relation to the time of delirium development. METHODS: Prospective cohort study in 13 ICUs from seven countries. Multiple logistic regression analysis was used to develop the early prediction (E-PRE-DELIRIC) model on data of the first two-thirds and validated on data of the last one-third of the patients from every participating ICU. RESULTS: In total, 2914 patients were included. Delirium incidence was 23.6%. The E-PRE-DELIRIC model consists of nine predictors assessed at ICU admission: age, history of cognitive impairment, history of alcohol abuse, blood urea nitrogen, admission category, urgent admission, mean arterial blood pressure, use of corticosteroids, and respiratory failure. The area under the receiver operating characteristic curve (AUROC) was 0.76 [95% confidence interval (CI) 0.73-0.77] in the development dataset and 0.75 (95% CI 0.71-0.79) in the validation dataset. The model was well calibrated. AUROC increased from 0.70 (95% CI 0.67-0.74), for delirium that developed <2 days, to 0.81 (95% CI 0.78-0.84), for delirium that developed >6 days. CONCLUSION: Patients' delirium risk for the complete ICU length of stay can be predicted at admission using the E-PRE-DELIRIC model, allowing early preventive interventions aimed to reduce incidence and severity of ICU delirium.

Comparison of direct and indirect radiation effects on osteoclast formation from progenitor cells derived from different hemopoietic sources.

Hemopoietic stem and progenitor cells from different sources differ in radiosensitivity. Recently, we have demonstrated that the multinucleated cell responsible for bone resorption and marrow cavity formation, the osteoclast, is in fact of hemopoietic lineage. In this investigation we have studied the radiosensitivity of osteoclast formation from two different hemopoietic tissues: fetal liver and adult bone marrow. Development of osteoclasts from hemopoietic progenitors was induced by coculture of hemopoietic cell populations with fetal mouse long bones depleted of their own osteoclast precursor pool. During culture, osteoclasts developed from the exogenous cell population and invaded the calcified hypertrophic cartilage of the long bone model, thereby giving rise to the formation of a primitive marrow cavity. To analyze the radiosensitivity of osteoclast formation, either the hemopoietic cells or the bone rudiments were irradiated before coculture. Fetal liver cells were found to be less radiosensitive than bone marrow cells. The D0, Dq values and extrapolation numbers were 1.69 Gy, 5.30 Gy, and 24.40 for fetal liver cells and 1.01 Gy, 1.85 Gy, and 6.02 for bone marrow cells. Irradiation of the (pre)osteoclast-free long bone rudiments instead of the hemopoietic sources resulted in a significant inhibition of osteoclast formation at doses of 4 Gy or more. This indirect effect appeared to be more prominent in the cocultures with fetal than with adult hemopoietic cells. Furthermore, radiation doses of 8.0-10.0 Gy indirectly affected the appearance of other cell types (e.g., granulocytes) in the newly formed but underdeveloped marrow cavity. The results indicate that osteoclast progenitors from different hemopoietic sources exhibit a distinct sensitivity to ionizing irradiation. Radiation injury to long bone rudiments disturbs the osteoclast-forming capacity as well as the hemopoietic microenvironment.

Radiation-induced changes in the cell membrane of cultured human endothelial cells.

We investigated the effect of irradiation on the kinetic characteristics of amino acid and glucose transport, and the effect on the activity of the cell membrane-bound enzyme 5'-nucleotidase and on the receptor-mediated stimulation of cyclic adenosine monophosphate synthesis by prostaglandin E1. Irradiation inhibited the sodium-dependent amino acid transport by a reduced binding of the amino acid to the transport unit. The transport of glucose, which appeared to be a sodium-independent process, was temporarily stimulated by increased maximal velocity of the transport. No effect was found on the binding to the transport unit. Irradiation increased the 5'-nucleotidase activity and decreased the prostaglandin E1-stimulated cyclic adenosine monophosphate synthesis 48 h after exposure to 20 Gy. It is concluded that irradiation decreases sodium-dependent transport by impairment of the transport unit, does not impair a sodium-independent process, and has opposite effects on membrane-bound enzyme activity and a receptor-mediated process.

An immunofluorescence assay for the detection of parvovirus B19 IgG and IgM antibodies based on recombinant viral antigen.

An indirect immunofluorescence assay for serum IgG and IgM antibodies to human parvovirus B19 was established using recombinant B19 viral antigen, the capsid protein VP1, which had been produced in a baculovirus expression system. This protein gives a strong and characteristic signal in the immunofluorescence assay, making it a suitable candidate for this test system. The test results showed a good correlation with results obtained with a solid-phase capture radioimmunoassay (Cohen et al., 1983). 76% of sera from a random selection of blood donors were positive for B19 IgG which agrees with previous findings. The course of the IgM and IgG antibody response to B19 infection could be followed with the immunofluorescence assay by determining the titers of series of sera taken after a recent B19 infection. Investigation of 24 sera containing rubella-specific IgM showed no cross-reactivity with the recombinant B19 VP1 used in this test system. The test described here has the advantage of being based on a renewable source of antigen and will be further evaluated for routine diagnostic use in comparison with radioimmunoassay.

Depressed responsiveness of peripheral blood mononuclear cells to heat-shock proteins in periodontitis patients.

The extensive homology between human and bacterial heat shock proteins (HSPs) may play a role in autoimmune reactions in periodontitis. Thus, we questioned whether peripheral blood mononuclear cell (PBMC) proliferative responses to HSPs are different between periodontitis patients and control subjects with gingivitis. The proliferative responses of PBMCs of patients (n = 10) and controls (n = 12) to recombinant mycobacterial HSP60 (MycHSP60) and HSP70 (MycHSP70), as well as recombinant human HSP60 (HumHSP60) and HSP70 (HumHSP70), were investigated. In addition, the proliferative responses to Candida albicans and purified protein derivatives of Mycobacterium (PPD) were included. Mean responses to HumHSP60, MycHSP60, and HumHSP70 were significantly lower for patients compared with controls. The responses to MycHSP70 showed a similar trend. However, when Candida and PPD were used as antigens, there was no difference in responses of the PBMCs between the periodontitis patients and controls. The level of IFN-gamma in the supernatants of the cells stimulated with HSPs was lower in the patients compared with controls. This concurs with the current hypothesis that periodontitis patients have a depressed Th1 response. Furthermore, we found that with an increasing estimated subgingival bacterial load, periodontitis patients mount a decreasing immune response to HSPs, while the controls showed a positive correlation between these two parameters. From these findings, we speculate that poor reactivity to HSPs may be a susceptibility factor for destructive periodontal disease and may need to be considered in the pathogenesis of this condition.

Osteoblasts develop from isolated fetal mouse chondrocytes when co-cultured in high density with brain tissue.

Chondrocytes from the hypertrophic and proliferative zones of 16-day-old fetal murine metatarsal bones were enzymatically dissociated and cultured in a high-density type of culture, exposed to the gas phase. We ascertained that no cells of the perichondrium were included in the cell suspension. Control cultures formed a solid cartilaginous mass, of which all the chondrocytes were alkaline phosphatase positive and the matrix started to calcify after 4 days. After 6 days, nearly the entire matrix was calcified. When co-cultured with pieces of cerebral tissue, some chondrocytes had transdifferentiated into osteoblasts after 4 days. They had started to form osteoid. After 6 and 11 days part of the cartilage had been replaced by bone, especially in the periphery of the cultures, but also in areas in the center. The bone matrix was partly calcified. Osteoblasts and bone matrix were identified as such electron microscopically. The nature of the bone matrix was also confirmed by immunohistochemical demonstration of collagen type I and osteocalcin. These results show that enzymatically isolated chondrocytes are able to become osteoblasts when properly stimulated. This supports the concept of chondrocytes being responsible for (part of) the endochondral bone formation in the marrow cavity of long bones.

Osteoblasts develop from isolated fetal mouse chondrocytes when co-cultured in high density with brain tissue.

Chondrocytes from the hypertrophic and proliferative zones of 16-day-old fetal murine metatarsal bones were enzymatically dissociated and cultured in a high-density type of culture, exposed to the gas phase. We ascertained that no cells of the perichondrium were included in the cell suspension. Control cultures formed a solid cartilaginous mass, of which all the chondrocytes were alkaline phosphatase positive and the matrix started to calcify after 4 days. After 6 days, nearly the entire matrix was calcified. When co-cultured with pieces of cerebral tissue, some chondrocytes had transdifferentiated into osteoblasts after 4 days. They had started to form osteoid. After 6 and 11 days part of the cartilage had been replaced by bone, especially in the periphery of the cultures, but also in areas in the center. The bone matrix was partly calcified. Osteoblasts and bone matrix were identified as such electron microscopically. The nature of the bone matrix was also confirmed by immunohistochemical demonstration of collagen type I and osteocalcin. These results show that enzymatically isolated chondrocytes are able to become osteoblasts when properly stimulated. This supports the concept of chondrocytes being responsible for (part of) the endochondral bone formation in the marrow cavity of long bones.

Proteolytic processing of the N-terminal region of the equine arteritis virus replicase.

A papainlike cysteine protease (PCP) domain in the N-terminal region of the equine arteritis virus (EAV) replicase was identified by in vitro translation and mutagenesis studies. The EAV protease was found to direct an autoproteolytic cleavage at its C-terminus which leads to the production of an approximately 30K N-terminal replicase product (nsp1) containing the PCP domain. Amino acid residues Cys164 and His230 of the EAV replicase polyprotein were identified as the most likely candidates for the role of PCP catalytic residues. It was shown that cleavage occurs in cis between Gly260 and Gly261.

Transdifferentiation of hypertrophic chondrocytes into osteoblasts in murine fetal metatarsal bones, induced by co-cultured cerebrum.

The fate of hypertrophic chondrocytes in 17-day-old metatarsal bones of fetal mice was studied in a culture system in which these cells were kept confined to their lacunae. Because the periosteum had been stripped off, osteoclasts could not invade the long bone and resorb the lacunar walls. The majority of the hypertrophic chondrocytes stayed alive and dedifferentiated gradually into cells with the appearance of stromal cells. When the long bones were co-cultured with pieces of cerebrum, the chondrocytes transdifferentiated into osteoblasts. We followed this process from day to day. The cells produced bone matrix that immunostained for collagen type I and osteocalcin. To exclude with certainty the possibility that the intralacunar osteoblasts had derived from remaining periosteal osteoprogenitor cells that invaded the lacunae, the long bones were pre-cultured with cytochalasin D, which inhibits cell proliferation and migration. After removal of the drug this effect persisted until after transdifferentiation had occurred. This proved that the bone matrix producing osteoblasts inside the cartilage lacunae were transdifferentiated chondrocytes. The transdifferentiation stimulating factor from brain tissue is still unknown.

Proteolytic processing of the replicase ORF1a protein of equine arteritis virus.

To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire ORF1a protein, results in cleavage products of approximately 29, 61, 22, 31, 41, and 3 kDa, which were named nonstructural proteins (nsps) 1 through 6, respectively. Pulse-chase experiments revealed that the cleavages at the nsp1/2 and nsp2/3 junctions are the most rapid processing steps. The remaining nsp3456 precursor is first cleaved at the nsp4/5 site. Final processing of the nsp34 and nsp56 intermediates is extremely slow. As predicted from previous in vitro translation experiments (E. J. Snijder, A. L. M. Wassenaar, and W. J. M. Spaan, J. Virol. 66:7040-7048, 1992), a cysteine protease domain in nsp1 was shown to be responsible for the nsp1/2 cleavage. The other processing steps are carried out by the putative EAV serine protease in nsp4 and by a third protease, which remains to be identified.

The 5' end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease.

The presence of a papainlike cysteine protease (PCP) domain in the N-terminal region of the equine arteritis virus (EAV) replicase, which had been postulated on the basis of limited sequence similarities with cellular and viral thiol proteases, was confirmed by in vitro translation and mutagenesis studies. The EAV protease was found to direct an autoproteolytic cleavage at its C terminus which leads to the production of an approximately 30-kDa N-terminal replicase product (nsp1) containing the PCP domain. Amino acid residues Cys-164 and His-230 of the EAV replicase polyprotein were identified as the most likely candidates for the role of PCP catalytic residues. By means of N-terminal sequence analysis of a PCP cleavage product, derived from a bacterial expression system, it was shown that cleavage occurs between Gly-260 and Gly-261. No evidence for PCP-directed cleavages at other positions in the EAV replicase was obtained. In cotranslational and posttranslational trans-cleavage assays, neither EAV nsp1 nor its precursor was able to process the PCP cleavage site in trans.

Type-1 and type-2 CD8+ T-cell subsets isolated from chronic adult periodontitis tissue differ in surface phenotype and biological functions.

Cloning of CD8+ T cells expressing the alpha beta T-cell receptor from inflamed human gingiva revealed that at least two different subsets were found within the tissue and that these subsets were able to interact with each other. One subset produced high levels of interferon-gamma (IFN-gamma) and no interleukin-4 (IL-4) or IL-5, exhibited phytohaemagglutinin (PHA)- or anti-CD3-mediated cytolytic activity, and were CD28+. The other subset produced high levels of IL-4 in combination with IL-5, displayed no cytotoxicity and were CD28-. From the latter subset CD8+ T-cell clones were able to suppress the proliferative response of cytotoxic CD8+ T-cell clones. This suppression could be abolished by anti-IL-4 monoclonal antibodies. However, IL-4 alone was not able to induce the suppression. Our results indicate that CD8+ T cells might participate in local immune responses by the suppression of IFN-gamma-producing cells and by favouring humoral responses via the production of IL-4 and IL-5.

The arterivirus Nsp2 protease. An unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases.

The replicase ORF1a polyprotein of equine arteritis virus, a positive-stranded RNA virus, is proteolytically processed into (at least) six nonstructural proteins (Nsp). A papain-like Cys protease in Nsp1 and a chymotrypsin-like Ser protease in Nsp4 are involved in this process. In this paper we demonstrate that the Nsp2/3 junction is not cleaved by either of these previously described proteases. Comparative sequence analysis suggested that an additional Cys protease resided in the N-terminal Nsp2 domain. For equine arteritis virus, this domain was shown to induce Nsp2/3 cleavage in a trans-cleavage assay. Processing was abolished when the putative active site residues, Cys-270 and His-332, were replaced. Other Nsp2 domains and three other conserved Cys residues were also shown to be essential. The Nsp2 Cys protease displays sequence similarity with viral papain-like proteases. However, the presumed catalytic Cys-270 is followed by a conserved Gly rather than the characteristic Trp. Replacement of Gly-271 by Trp abolished the Nsp2/3 cleavage. Conservation of a Cys-Gly dipeptide is a hallmark of viral chymotrypsin-like Cys proteases. Thus, the arterivirus Nsp2 protease is an unusual Cys protease with amino acid sequence similarities to both papain-like and chymotrypsin-like proteases.

Arrangement of integrated viral DNA sequences in cells transformed by adenovirus types 2 and 5.

The organization of the viral DNA sequences in 15 adenovirus-transformed cell lines was analyzed by the Southern blotting procedure. The site of adenovirus integration in the cellular genome was found not to be unique, and the viral DNA sequences involved in integration were not confined to a specific region of the adenovirus genome. Several cell lines showed simple integration patterns that demonstrated the presence of large continuous stretches of viral DNA. In four cell lines, containing sequences from both molecular ends of the viral genome, the left- and right-hand-terminal sequences appeared to be linked to each other.

Alkaline phosphatase and calcification, correlated or not?

The effects of alkaline phosphatase inhibitors (levamisole, L-bromotetramisole) on the activity of the enzyme and on calcification in vitro were studied, to find out whether there is a relationship between alkaline phosphatase and calcification. Metatarsal bones of 15 1/4-day-old embryonic mice were dissected and cultured for 40 hours in the presence and absence of inhibitor. Levamisole and L-bromotetramisole fully inhibited calcification in vitro when present in concentrations which almost totally inhibited alkaline phosphatase activity, as measured biochemically or histochemically. However, incorporation of 3H-thymidine and 35S-sulphate was also inhibited. Furthermore, D-bromotetramisole, the dextroform of bromotetramisole which has no effect on alkaline phosphatase, inhibited calcification and 3H-thymidine and 35S-sulphate incorporation as well. The results of this study show that these inhibitors cannot be used to study the relationship between alkaline phosphatase and calcification. In addition, they suggest that although alkaline phosphatase may be important for the process of calcification, it is probably not a critical factor.