Employment status 1 year after out-of-hospital cardiac arrest in comatose patients treated with therapeutic hypothermia.

BACKGROUND: Therapeutic hypothermia for comatose survivors of out-of-hospital cardiac arrest (OHCA) has improved survival and neurologic outcome. This study focused on return to work 1 year after therapeutic hypothermia. METHODS: From June 2004 to June 2009, patients between 18 and 65 years of age with OHCA, who were treated with hypothermia from two regions, representing one third of the national population, were identified from the Danish National Patient Registry, and from hospital and ambulance records. The patients' employment status was obtained from the Danish Ministry of Employment. RESULTS: One hundred thirty-three comatose patients after OHCA treated with hypothermia were identified. One hundred and four (78%) patients were employed, or able to work, at the time of cardiac arrest. This particular group of patients showed significant lower in-hospital mortality compared to the group of patients who were not able to work before cardiac arrest; 13% vs. 48%, respectively (P < 0.001). The workable group had a lower Charlson comorbidity score (P = 0.004), a higher incidence of witnessed cardiac arrest (P = 0.004) and a higher incidence of shockable heart rhythm (P < 0.001). Eighty-seven patients (84%), who were able to work prior to cardiac arrest, survived, and 55 (65%) of these patients were employed or able to work at 1 year follow-up. CONCLUSION: The majority of patients employed, or able to work prior to OHCA, had returned to work at one year follow-up. Predictors of return to work in comatose patients treated with hypothermia have to be identified in a larger-scale study.

Synchrotron vacuum ultraviolet radiation studies of the D (1)Π(u) state of H(2).

The 3pπD (1)Π(u) state of the H(2) molecule was reinvestigated with different techniques at two synchrotron installations. The Fourier transform spectrometer in the vacuum ultraviolet wavelength range of the DESIRS beamline at the SOLEIL synchrotron was used for recording absorption spectra of the D (1)Π(u) state at high resolution and high absolute accuracy, limited only by the Doppler contribution at 100 K. From these measurements, line positions were extracted, in particular, for the narrow resonances involving (1)Π(u) (-) states, with an accuracy estimated at 0.06 cm(-1). The new data also closely match multichannel quantum defect calculations performed for the Π(-) components observed via the narrow Q-lines. The Λ-doubling in the D (1)Π(u) state was determined up to v=17. The 10 m normal incidence scanning monochromator at the beamline U125/2 of the BESSY II synchrotron, combined with a home-built target chamber and equipped with a variety of detectors, was used to unravel information on ionization, dissociation, and intramolecular fluorescence decay for the D (1)Π(u) vibrational series. The combined results yield accurate information on the characteristic Beutler-Fano profiles associated with the strongly predissociated Π(u) (+) parity components of the D (1)Π(u) levels. Values for the parameters describing the predissociation width as well as the Fano-q line shape parameters for the J=1 and J=2 rotational states were determined for the sequence of vibrational quantum numbers up to v=17.

Massive upper GI bleeding: a rare complication of Zenker's diverticulum.

Bleeding from a Zenker's diverticulum is rare. A 71-year-old man was urgently admitted with massive hematemesis. It was known that he had a Zenker's diverticulum, but on emergency endoscopy, the source of bleeding was not detected due to large blood clots in the esophagus, hypo-pharynx and also into the tracheal-bronchial tree. Computerized tomography angiography demonstrated a blush of intravenous contrast arising from the diverticulum. The patient was operated upon urgently; the diverticle had a deep ulceration which was the source of the bleeding. The cause of the ulceration is unknown but it is possible that it was caused by the direct effect of an aspirin pill within the diverticle. A similar case with the same conclusion has been published in the past and since the use of aspirin has become common, especially in the elder population, we present this case report to highlight this possible life-threatening complication of Zenker's diverticulum in patients receiving aspirin.

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays.

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8(+) T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

Therapeutic drug monitoring of the HIV/AIDS drugs abacavir, zidovudine, efavirenz, nevirapine, indinavir, lopinavir, and nelfinavir.

Combination therapy with antiretroviral drugs is used for the treatment of patients infected with the human immunodeficiency virus. To achieve optimal drug concentrations for viral suppression and avoidance of drug toxicity, monitoring of drug levels has been considered essential. We set up an analytical procedure for monitoring the plasma concentrations of a total of seven drugs: abacavir, zidovudine, efavirenz, nevirapine, indinavir, lopinavir, and nelfinavir. The plasma samples were liquid/liquid extracted and subjected to high-performance liquid chromatography (HPLC) analysis. The compounds were monitored by ultraviolet detection: indinavir, lopinavir, and nelfinavir at 215 nm; efavirenz at 254 nm, and abacavir, zidovudine, and nevirapine at 266 nm. Two different extraction procedures and two different HPLC eluents on a C(8) reversed-phase HPLC column were used to monitor all seven compounds. Under steady state conditions, the plasma concentrations of antiviral drugs in 175 patients were correlated with the time after the last dosing to define the peak or trough levels. Due to the short plasma elimination half-life of abacavir and zidovudine, only peak levels could be determined for these compounds, whereas both peak and trough levels could be assessed for the other compounds because of a longer plasma elimination half-life. The mean peak concentrations (microg/ml) were 0.69 for abacavir and 0.57 for zidovudine; the mean peak/trough concentrations (microg/ml) were 2.07/1.32 for efavirenz, 2.43/2.23 for nevirapine, 5.48/1.08 for indinavir, 4.69/3.51 for lopinavir, and 3.54/1.45 for nelfinavir. The described analytical method offers a broad-spectrum monitoring of plasma levels of antiretroviral drugs.

Does sophisticated diagnostic workup on neuroectodermal tumors have an impact on the treatment of esthesioneuroblastoma?

BACKGROUND: The diagnostic workup on esthesioneuroblastoma is more extensive than ever before. We have investigated whether improvements in diagnosis of sinonasal neuroectodermal tumors, including esthesioneuroblastomas (ENB), sinonasal neuroendocrine carcinomas (SNEC) and sinonasal undifferentiated carcinomas (SNUC), have had an impact on treatment and outcome. PATIENTS AND METHODS: 11 ENB, 7 SNEC and 1 SNUC in 13 men and 6 women (average age 52.9 years (range 26-82)), diagnosed between 1986 and 2001, were analyzed with regard to histopathologic and clinical diagnosis as well as outcome. Our results were compared with the available literature. RESULTS: According to the Morita classification considering endoscopy, CT and MRI scans, 2 tumors were staged D, 14 were found to be stage C, 2 were stage B and 1 was stage A. Lightmicroscopically only 4 of 19 showed higher differentiation and rosette-like structures, the others were poorly differentiated. 18 of 19 tumors were examined immunohistochemically. Neuronal markers (NSE, synaptophysin, chromogranin, S-100 and neurofilaments) were heterogeneously expressed in both ENB and NEC, only NSE stained all but 2 tumors. Coexpression of neuronal markers and cytokeratins was proven in all NEC and 5 of 11 ENB. Some tumors expressed atypical markers. Despite extensive diagnostic steps it was not possible to exclude a different histopathological diagnosis in 10 of 19 cases. CONCLUSION: For sinonasal neuroectodermal tumors no pathognomonic antigenic profiles are known. Immunohistochemical markers lack specificity and sensitivity. Nevertheless, in many sinonasal neuroectodermal tumors a panel of differentiation markers allows to specify the light-microscopic diagnosis. Until now no therapeutic consequence arises from a more extensive diagnostic workup. However, the histopathologic identification of subtypes (SNUC) and proliferation markers may help to identify patients with poor prognosis.

Glutathione deficiency of the lower respiratory tract in patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown aetiology. Increased oxidant burden and antioxidant, e.g. glutathione (GSH), deficiency in the lower respiratory tract have been thought to play a role in the progression of IPF. Sputum induction is a safe noninvasive tool to study inflammation in the respiratory tract. The aim of the present study was to evaluate the direct measurement of GSH in induced sputum supernatant. Sixteen IPF patients and 15 healthy, nonsmoking subjects underwent sputum induction. Total GSH in sputum, saliva and plasma was measured spectrophotometrically. Sputum GSH was decreased more then four-fold in IPF patients when compared to healthy subjects (mean GSH 1.4+/-0.34 microM versus 5.8+/-0.98 microM). Salivary GSH was generally low or undetectable in all subjects. Plasma GSH levels were lower in IPF patients (0.26+/-0.1 versus 0.74+/-0.16 microM). In IPF patients, there was a borderline correlation of sputum GSH levels with disease duration and lung-function impairment. These data confirm the established role of oxidant/antioxidant imbalance in the pathogenesis of idiopathic pulmonary fibrosis, and show the potential of induced sputum to directly study inflammatory processes and surrogate markers in interstitial lung diseases like idiopathic pulmonary fibrosis.

The dilemma of follow-up in head and neck cancer patients.

The aims of tumor follow-up in head and neck cancer patients are (1) evaluation of therapeutic efficacy, (2) management of impairments, (3) detection of new tumor manifestations, and (4) psychosocial care. In general standardized 5-year-protocols are used for all such patients. However, it is questionable whether a rigid follow-up schedule is optimal for a very heterogeneous tumor population. Therefore 603 patients with sqamous cell carcinoma of the oral cavity, pharynx or larynx, or with cervical metastasis from an unknown primary site (CUP syndrome), who had been diagnosed and treated curatively by an operation with or without radiotherapy (n = 523) or just by radio(chemo)therapy (n = 80) between 1985 and 1994, and who had been followed-up regularly according to a standardized plan, were worked-up retrospectively. Data were evaluated for the manifestation and prognosis of curable new tumor manifestations as well as for tumor-specific factors likely to select groups which should be followed more or less intensively. Within a 5-year follow-up period new tumor growth was detected in 152/603 (25%) patients: 79 local and 31 regional recurrences, 18 systemic metastases and 24 second primary cancers. Where follow-up was extended beyond the 5th year, 168/603 (28%) patients presented a new tumor manifestation. One hundred and sixteen of the 152 (28%) patients had another operation with or without radiotherapy or had radio(chemo)therapy alone. So far 18/116 (14%) patients have survived their new tumor manifestation for more than 5 years and 30/116 for more than 2 years. Tumor-specific data on the initial tumors (T stage, N stage, site) did not indicate the risk of a new tumor manifestation, but 87% of patients who survived their new tumor manifestation for more than 2 years initially had T1 or T2 tumors and only 30% initially had N+ necks. Occurrence of distant metastasis or a second primary outside the head and neck region limited survival to < or = 2 years after detection. In terms of survival, follow-up efforts should therefore concentrate on detection of locoregional recurrence, particularly if an option for further curative local therapy exists. The limited success of detection of new tumor manifestations in terms of survival does not justify a reduction in tumor-follow-up examinations, since the benefit of the other efforts cannot be determined from survival figures.

Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes.

Cotranslational protein transport into dog pancreas microsomes involves the Sec61p complex plus a luminal heat shock protein 70. Posttranslational protein transport into the yeast endoplasmic reticulum (ER) involves the so-called Sec complex in the membrane, comprising a similar Sec61p subcomplex, the putative signal peptide receptor subcomplex, and the heat shock protein 40-type subunit, Sec63p, plus a luminal heat shock protein 70. Recently, human homologs of yeast proteins Sec62p and Sec63p were discovered. Here we determined the concentrations of these two membrane proteins in dog pancreas microsomes and observed that the canine homologs of yeast proteins Sec62p and Sec63p are abundant proteins, present in almost equimolar concentrations as compared with Sec61alphap monomers. Furthermore, we detected fractions of these two proteins in association with each other as well as with the Sec61p complex. The J domain of the human Sec63p was shown to interact with immunoglobulin heavy chain binding protein. Thus, the membrane of the mammalian ER contains components, known from the posttranslationally operating protein translocase in yeast. We suggest that these components are required for efficient cotranslational protein transport into the mammalian ER as well as for other transport processes.

Dissociation from BiP and retrotranslocation of unassembled immunoglobulin light chains are tightly coupled to proteasome activity.

Unassembled immunoglobulin light chains expressed by the mouse plasmacytoma cell line NS1 (kappa(NS1)) are degraded in vivo with a half-life of 50-60 min in a way that closely resembles endoplasmic reticulum (ER)-associated degradation (). Here we show that the peptide aldehydes MG132 and PS1 and the specific proteasome inhibitor lactacystin effectively increased the half-life of kappa(NS1), arguing for a proteasome-mediated degradation pathway. Subcellular fractionation and protease protection assays have indicated an ER localization of kappa(NS1) upon proteasome inhibition. This was independently confirmed by the analysis of the folding state of kappa(NS1) and size fractionation experiments showing that the immunoglobulin light chain remained bound to the ER chaperone BiP when the activity of the proteasome was blocked. Moreover, kinetic studies performed in lactacystin-treated cells revealed a time-dependent increase in the physical stability of the BiP-kappa(NS1) complex, suggesting that additional proteins are present in the older complex. Together, our data support a model for ER-associated degradation in which both the release of a soluble nonglycosylated protein from BiP and its retrotranslocation out of the ER are tightly coupled with proteasome activity.

Mannosidase action, independent of glucose trimming, is essential for proteasome-mediated degradation of unassembled glycosylated Ig light chains.

In order to study the role of N-glycans in the ER-associated degradation of unassembled immunoglobulin light (Ig L) chains, we introduced N-glycan acceptor sites into the variable domain of the murine Ig L chain kappaNS1, which is unfolded in unassembled molecules. We investigated the fate of kappaNS1 glycosylated at position 70 (K70) and of a double mutant (kappa18/70) in stably transfected HeLa cells. Degradation of both chains was impaired by lactacystin, a specific inhibitor of the proteasome. The mannosidase inhibitor dMNJ also blocked degradation in a step preceding proteasome action, as did two protein synthesis inhibitors, cycloheximide and puromycin. In contrast, ER glucosidase inhibitors dramatically accelerated the degradation of the chains when added either pre- or posttranslationally. The accelerated degradation was sensitive to lactacystin, dMNJ and cycloheximide, too. None of these drugs, except lactacystin, affected the degradation of unglycosylated kappaNS1 chains. We conclude that ER mannosidases and proteasome activities, but not glucose trimming (and therefore, most likely not the calnexin/calreticulin UDP:glucose glycoprotein glucosyl transferase cycle), are essential for ER-associated degradation (ERAD) of soluble glycoproteins. A role for a short-lived protein, acting before or simultaneously to ER mannosidases, is suggested.

Molecular characterization of a novel mammalian DnaJ-like Sec63p homolog.

We identified a human cDNA sequence encoding a polypeptide of 760 amino acids that shares 53% homology and 25.6% identity with the yeast DnaJ-like endoplasmic reticulum (ER) translocon component Sec63p. Three epitope-specific antisera revealed a protein of an apparent molecular mass of 83 kDa, both in human cell extracts and in dog pancreatic microsomes. Biochemical analyses show that it is an integral membrane protein of the rough ER, which has the DnaJ domain located in the ER lumen. The novel Sec63 protein could thus represent a key component of the mammalian ER protein translocation machinery.

Cycloheximide, a new tool to dissect specific steps in ER-associated degradation of different substrates.

To study the degradation requirements of unassembled immunoglobulin (Ig) chains, we heterologously expressed a cDNA encoding the secretory form of murine mu in the yeast S. cerevisiae. We found that mu chains were translocated into and retained in the endoplasmic reticulum (ER) as they were N-glycosylated and bound to the yeast homolog of BiP, Kar2p. Similar to mutant yeast carboxypeptidase Y (CPY*), known to undergo cytosolic degradation, mu protein is stabilized in yeast mutants lacking the ubiquitinating enzymes Ubc6p and Ubc7p or in cells overexpressing mutant ubiquitin. Unexpectedly, the translation inhibitor cycloheximide (CHX), but not puromycin, led to the accumulation of polyubiquitinated mu chains that were still glycosylated. By contrast, degradation of CPY* was not impaired by CHX, indicating that the drug affects a substrate-specific degradation step. In contrast to the situation for CPY*, the ER-transmembrane protein Der1p is not essential for mu degradation. Strikingly, however, the CHX-induced accumulation of polyubiquitinated Igmu chains was stronger in deltader1-mutants as compared to wild-type cells, indicating an additive effect of two inhibitory conditions. The results support a previously unknown activity of CHX, i.e. impairing the degradation of transport-incompetent secretory mu chains. Moreover, this activity will allow to dissect substrate-specific steps in ER associated protein degradation.

Anti-(epidermal growth factor) receptor monoclonal antibodies for the induction of antibody-dependent cell-mediated cytotoxicity against squamous cell carcinoma lines of the head and neck.

Squamous cell carcinomas of the head and neck (SCCHN) frequently display high levels of the epidermal growth factor receptor (EGFR). Since EGFR is expressed on the cell surface it may form a suitable target for anticancer therapy with anti-receptor monoclonal antibodies (mAb). Besides the interference with receptor/ligand interactions, binding of mAb to EGFR leads to immunoglobulin-coated tumour cells that may induce or enhance non-specific immune effector mechanisms like antibody-dependent cell-mediated cytotoxicity (ADCC). In established cell lines of SCCHN (UM-SCC 11B, 14C, 22B, and 8029 NA) we investigated the antitumour activity of allogeneic peripheral blood mononuclear cells (PBMC) in combination with rat (ICR 62), mouse (EMD 55900), and humanized (EMD 72000) anti-EGFR mAb. In addition, autologous PBMC were available for tumour line UD-SCC 4. The EGFR protein content of the tumour cell lines ranged between 170 fmol/mg protein and 8100 fmol/mg protein, and MCF-7 cells served as receptor-negative controls. PBMC activity against SCCHN targets was determined in 96-well microtitre-plate monolayer cultures by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after coincubation for 4 h, 24 h and 72 h at effector target ratios of 1:1, 5:1, 10:1 and 20:1. PBMC subpopulations were obtained by macrophage depletion (plastic adherence) or natural killer (NK) cell enrichment (magnetic bead negative selection). Prolonged time of exposure and increased effector:target ratios revealed marked antitumour activity of PBMC alone. This non-specific immune destruction was enhanced considerably by humanized and rat, but not mouse anti-EGFR mAb. Increased EGFR protein in tumour cells partly correlated with an intensification of ADCC but was accompanied by decreased primary PBMC cytotoxicity. The utilization of PBMC subpopulations suggested a mainly NK-cell-mediated ADCC, which appeared to benefit directly or indirectly, e.g. via the secretion of cytokines, from other PBMC components. In conclusion, humanized (EMD 72000) and rat (ICR 62) anti-EGFR mAb were able to generate strong antitumour ADCC in target monolayers of SCCHN.

The variable domain of nonassembled Ig light chains determines both their half-life and binding to the chaperone BiP.

Not much is known about the features that determine the biological stability of a molecule retained in the endoplasmic reticulum (ER). Ig light (L) chains that are not secreted in the absence of Ig heavy (H) chain expression bind to the ER chaperone BiP as partially folded molecules until they are degraded. Although all Ig L chains have the same three-dimensional structure when part of an antibody molecule, the degradation rate of unassembled Ig L chains is not identical. For instance, the two nonsecreted murine Ig L chains, kappaNS1 and lambdaFS62, are degraded with half-lives of approximately 1 and 4 hr, respectively, in the same NS1 myeloma cells. Furthermore, the BiP/lambdaFS62 Ig L chain complex appears to be more stable than the BiP/kappaNS1 complex. Here, we used the ability of single Ig domains to form an internal disulfide bond after folding as a measure of the folding state of kappaNS1 and lambdaFS62 Ig L chains. Both of these nonsecreted L chains lack the internal disulfide bond in the variable (V) domain, whereas the constant (C) domain was folded in that respect. In both cases the unfolded V domain provided the BiP binding site. The stability of BiP binding to these two nonsecreted proteins was quite different, and both the stability of the BiP:Ig L chain complex and the half-life of the Ig L chain could be transferred from one Ig L chain isotype to the other by swapping the V domains. Our data suggest that the physical stability of BiP association with an unfolded region of a given light chain determines the half-life of that light chain, indicating a direct link between chaperone interaction and delivery of partially folded substrates to the mammalian degradation machinery.

Differential fate of glycoproteins carrying a monoglucosylated form of truncated N-glycan in a new CHO line, MadIA214214, selected for a thermosensitive secretory defect.

A temperature sensitive secretory line, MadIA214, was selected from mutagenized Chinese hamster ovary cells that express two heterologous export marker proteins: a secretory form of the human placental alkaline phosphatase (SeAP), and the Kd heavy chain of mouse MHC class I. SeAP secretion in MadIA214 was extremely reduced at elevated temperature (40 degrees C), while the export of functional H-2Kd molecules to the plasma membrane was only slightly affected. This mutant constitutively transferred onto newly synthesized proteins a truncated oligosaccharide core, Man5GlcNAc2, which was monoglucosylated in the protein-bound form. Nevertheless, the final oligosaccharide-structures associated to mature SeAP and H-2Kd were similar in mutant and wild-type glycoproteins. The inaccessibility in MadIA214 endoplasmic reticulum (ER) of one or more components required for oligosaccharide chain elongation is supported by the reconstitution of a correct core structure, obtained after disruption of cellular compartments, but not after cell permeabilisation or blocking ER-to-Golgi transport. The increased association of the ER-chaperone BiP with immature SeAP correlated with the thermodependent decrease in SeAP secretion. The retention of incompletely folded polypeptides in MadIA214 parallels both a marked ER-dilation and an important glycoprotein degradation documented by the formation of soluble oligomannosides with one GlcNAc residue. Our data provide the first in vivo evidence that the initial step in N-glycosylation differentially governs glycoprotein maturation, transport and degradation.

Assembly of immunoglobulin light chains as a prerequisite for secretion. A model for oligomerization-dependent subunit folding.

Oligomeric proteins usually have to assemble into their final quartenary structure to be secreted. However, most immunoglobulin (Ig) light (L) chains can be exported as free chains, whereas only a few Ig L chains, here referred to as export-incompetent, have to assemble with Ig heavy (H) chains into antibody molecules to be secreted. In the absence of Ig H chain expression, these export-incompetent Ig L chains remain bound to BiP as partially folded monomers with only one of the two internal disulfide bonds being formed. To understand the apparent discrepancy in Ig L chain export, we performed assembly studies with chimeric Ig chains and found that the variable (V) domain of the export-incompetent NS1 kappa chain cannot mediate homodimer formation. Conversely, the V domain of the export-competent J558L lambda1 chain supports homodimer formation and, concordantly, these Ig L chains are secreted as noncovalently or covalently linked homodimers. We show that the export-incompetent mutant lambda1 FS62 chain forms disulfide bonds in both domains only upon pairing with Ig H chain and is secreted as part of an antibody. Therefore, Ig L chain assembly seems to be a prerequisite for complete folding, indicating that Ig L chain secretion generally depends on either homo- or heterodimer formation. We discuss a mechanism that controls oligomerization by monitoring the conformation of individual subunits that cannot proceed in folding prior to successful assembly.