bluuebunny/crossref_metadata_2025 huggingface dataset:
Fatigue vs. shortening‐induced deactivation in striated muscle
Authors: EDMAN K. A. P.
Published: November 2003

Fatigue and shortening‐induced deactivation, two conditions that both lead to reversible depression of the mechanical performance of striated muscle are briefly reviewed.

Fatigue. Isolated fibres from frog skeletal muscle (1–3 °C) that are stimulated to produce a 1 s fused tetanus at 15 s intervals are brought into a state of reduced to 70–75% of the control) that is attributable to reduced performance of the myofibrils with no significant change in activation of the contractile system. A more intense stimulation programme (a single stimulus applied at 1–2 s intervals) reduces the tetanic force below 70% of the rested‐state level. Under these conditions, failure of activation becomes increasingly important as a cause of the force decline. Deficient inward spread of activation is likely to account for at least part of the force decline after a period of intense fatiguing stimulation.

Shortening‐induced deactivation. Striated muscle that is allowed to shorten during activity loses some of its capacity to produce force, full restoration of the contractile strength being attained 1–2 s after the shortening phase. The depressant effect of shortening is demonstrable in skinned preparations as well as in intact muscle fibres and the magnitude of the effect is dependent on the state of activation of the muscle fibre when the movement occurs. The experimental evidence supports the view that sliding of the thick and thin filaments during activity reduces the affinity for calcium at the regulatory sites on the thin filament, leading to a transitory deactivation of the contractile system.
Treadmill exercise in mice increases intestinal lymphocyte loss via apoptosis
Authors: Hoffman‐Goetz L. , Quadrilatero J.
Published: November 2003

Strenuous exercise is associated with a transient decline in circulating lymphocytes, possibly through increased apoptosis. Intestinal lymphocytes are important effector cells of intestinal immune function but have not been studied in relation to exercise.

Aim:  The purpose of the present study was to examine the effect of exercise on intestinal lymphocyte phenotypes and apoptosis.

Methods:  Female C57BL/6 mice (n = 112) were randomized to: (1) treadmill running (90 min, 32 m min−1, 8° grade) and killed immediately after exercise, (2) treadmill running and killed 2 h after exercise, (3) treadmill running and killed 24 h after exercise or (4) a non‐exercised control condition with exposure to treadmill noise and vibration without running.

Results:  Flow cytometry indicated that the total intestinal CD3+T (P < 0.01), CD4+T (P < 0.005), CD8+T (P < 0.05), pan‐NK (P < 0.005) and CD19+B (P < 0.05) lymphocytes were significantly lower 24 h after exercise compared with non‐exercised controls. Significantly more CD3+T (P < 0.05) and CD8+T (P < 0.05) intestinal lymphocytes stained positive for annexin V, a marker of apoptosis, at 24 h after exercise compared with intestinal lymphocytes from non‐exercised controls. Plasma corticosterone and 8‐isoprostane concentrations were also significantly higher immediately after exercise compared with other exercise conditions.

Conclusion:  Acute strenuous exercise increases intestinal T (CD3+ and CD8+) lymphocyte loss and apoptosis. The extent to which the exercise‐induced apoptosis in intestinal lymphocytes is mediated by increased glucocorticoid concentrations in the gastrointestinal tract will require further studies.
Artemisinin population pharmacokinetics in children and adults with uncomplicated falciparum malaria
Authors: Sidhu J. S. , Ashton M. , Huong N. V. , Hai T. N. , Karlsson M. O. , Sy N. D. , Jonsson E. N. , Cong L. D.
Published: November 2003

Aims
 To investigate the pharmacokinetics of the antimalarial artemisinin in the field setting using sparsely collected data.

Methods
 Artemisinin concentrations were determined by h.p.l.c. in a total of 107 capillary plasma samples collected on the first day and in 33 samples on the last day of a 5‐day oral artemisinin regimen of 10 mg kg−1 day−1 in 23 paediatric (aged 2–12 years) and 31 adult (aged 16–45 years) Vietnamese patients with uncomplicated falciparum malaria. The population model was developed using NONMEM, incorporating interoccasion variability and accounting for a systematic change in artemisinin pharmacokinetics with time, modelled as a change in oral bioavailability.

Results
 Clinical efficacy, in terms of parasite clearance and fever subsidence times, was comparable between children and adults. A one‐compartment model with separate pharmacokinetic estimates for children and adults was found best to describe the disposition of artemisinin after oral administration. The population estimates for artemisinin clearance and distribution volume, respectively, were 432 l h−1 and 1600 l for adults and 14.4 l h−1 kg−1 and 37.9 l kg−1 for children, with an intersubject variability (collectively for both age groups) of 45% and 104%, respectively. The oral bioavailability was estimated to decrease from Day 1 to Day 5 by a factor of 6.9, a value found to be similar for children and adults.

Conclusions
 Artemisinin pharmacokinetic data was successfully derived in both paediatric and adult patients using 2–3 capillary blood samples taken in conjunction with parasitaemia monitoring. This study’s findings advocated the dosing of artemisinin to children according to bodyweight and to adults according to a standard dose.
Effect of selective phosphodiesterase 3 inhibition on the early and late asthmatic responses to inhaled allergen
Authors: Bardin Philip G. , Dorward Martina A. , Lampe Fiona C. , Franke Brigitte , Holgate Stephen T.
Published: November 2003

Aims
 Phosphodiesterase isoenzymes may play an important role in the regulation of airway calibre and bronchial smooth muscle function. Immunomodulatory actions may also be important in allergic airway inflammation. We have examined the effect of a phosphodiesterase (PDE) 3 inhibitor (MKS492) on the early and late responses to inhaled allergen in 18 atopic asthmatic subjects.

Methods
 On three separate occasions, 2–4 weeks apart, patients were administered either placebo, 20 mg or 40 mg of oral MKS492 as a single dose. After 90 min they inhaled a dose of allergen (grass pollen or D. pteronyssinus ) that had been demonstrated to produce a 20% decrease in FEV1. Measurements of FEV1 were repeated at 30 min intervals over the subsequent 7.5 h.

Results
 After placebo, allergen inhalation produced bronchoconstriction with a maximum mean minimum FEV1 of 75.9% (12.7) (mean [s.d.]) of post‐saline baseline values and a late response developed in 13 subjects (decrease in FEV1≥15%). MKS492 (40 mg) increased the mean minimum % post‐saline FEV1 at 30 min from 81.6% (14.0) for placebo to 93.1% (13.8), difference 11.5, 95% CI (2.6, 20.4). The mean minimum % post‐saline FEV1 in the early response (0‐2 h) was also increased by the 40 mg dose to 86.5% (11.8) vs 77.9% (13.8) for placebo, but not by the 20 mg dose. Significant bronchodilatation was noted 90 min after dosing (pre‐allergen) in the MKS492 40 mg group. Late phase responses as assessed by 4 h % post‐saline FEV1 was significantly improved by MKS 492 40 mg to 90.0% (6.3) vs 83.0% (12.4) for placebo–difference 7.0, 95% CI (0.1, 13.9), but again not by the 20 mg dose. Analysis of the area under the FEV1 response‐time course curves showed non‐significant reduction in the late response after the 40 mg dose (placebo 159.0, 134.8) vs 128.1, [81.5] for 40 mg).

Conclusions
 A novel PDE 3 inhibitor significantly decreases the early bronchoconstrictor response in asthma and attenuates the late response; effects that may be more marked at higher doses.
The airway effects of stopping regular oral theophylline in patients with asthma
Authors: Bennett J. A. , Thompson Coon J. , Pavord I. D. , Wilding P. J. , Tattersfield A. E.
Published: November 2003

Aims
 We investigated whether the deterioration in asthma control reported following cessation of theophylline was due to tolerance to theophylline.

Methods
 Eighteen subjects with mild stable asthma were given oral theophylline 10 mg kg−1 day−1 or placebo for 2 weeks in a double‐blind crossover study. FEV1 and PD20 histamine were measured before and 8 h after the first dose of treatment and 8, 32 and 56 h after the final dose. PD20 AMP was measured before treatment and 9 h after the final dose.

Results
 Six patients did not tolerate theophylline. In the other 12 subjects there were no differences between treatments in daily PEF, symptom scores, rescue bronchodilator use, PD20 histamine or FEV1 up to 8 h post treatment. Following withdrawal of theophylline there were significantly lower values for mean FEV1 (mean difference 0.15 l, 95% CI 0.03, 026) and PD20 AMP compared to placebo but no difference in other end points.

Conclusions
 The small rebound deterioration in lung function following regular treatment with therapeutic doses of oral theophylline is consistent with the development of tolerance.
Inhibitory effects of antiarrhythmic drugs on phenacetin O‐deethylation catalysed by human CYP1A2
Authors: Kobayashi K. , Nakajima M. , Chiba K. , Yamamoto T. , Tani M. , Ishizaki T. , Kuroiwa Y.
Published: November 2003

Aims
 The aim of the study was to clarify whether the pharmacokinetic interaction between theophylline and mexiletine is mediated by inhibition of CYP1A2 and to assess the possible interaction potential of other antiarrhythmic drugs with drugs metabolized by CYP1A2.

Methods
 The inhibitory effects of mexiletine and 10 antiarrhythmic drugs on phenacetin O‐deethylation, a marker reaction of CYP1A2, were studied using human liver microsomes and cDNA‐expressed CYP1A2.

Results
 Propafenone and mexiletine inhibited phenacetin O‐deethylation with IC50 values of 29 and 37 μm, respectively. Disopyramide, procainamide and pilsicainide produced negligible inhibition of phenacetin O‐deethylation (IC50>1 mm ). Amiodarone, bepridil, aprindine, lignocaine, flecainide and quinidine inhibited phenacetin O‐deethylation in a concentration‐dependent manner, although the inhibitory effects were relatively weak with IC50 values ranging from 86 to 704 μm. Propafenone and mexiletine selectively abolished the high‐affinity component of phenacetin O‐deethylation in human liver microsomes. In addition, propafenone and mexiletine inhibited phenacetin O‐deethylation catalysed by cDNA‐expressed CYP1A2.

Conclusions
 These data suggest that, among the antiarrhythmic drugs studied, propafenone and mexiletine are relatively potent inhibitors of CYP1A2, which may cause a drug‐drug interaction with drugs metabolized by CYP1A2.
Vigabatrin:placental transfer ‘ooin vivo‘ox and excretion into breast milk of the enantiomers
Authors: Tran A. , O’Mahoney T. , Rey E. , Mai J. , Mumford J. P. , Olive G.
Published: November 2003

Aims
 Vigabatrin is a new antiepileptic medication consisting of a racemic mixture of 50% active S enantiomer and 50% inactive R enantiomer. Since patients suffering from epilepsy may become pregnant, it is important to understand the extent of placental transfer of such medication.

Methods
 During steady‐state, vigabatrin enantiomer concentrations were measured in maternal and umbilical blood and in breast milk of two patients.

Results
 The concentration ratios from the umbilical vein to maternal plasma were R:0.068, S:0.16; 4h25 min after drug administration (case 1) and R:1.39, S:0.91; 9h after drug administration (case 2). The milk:plasma concentration ratio was lower than 1 at pre dose sampling in both cases, as well as 3 and 6 h post dose in one case. An estimate of the maximum amount of R and S enantiomers of vigabatrin that a suckling infant would ingest in a day is 3.6% and 1% of the weight‐adjusted daily dose respectively.

Conclusions
 These results would suggest a slow placental transfer of the vigabatrin enantiomers and that the quantity ingested through milk is small.
Single dose and steady state pharmacokinetics and pharmacodynamics of the ACE‐inhibitor imidapril in hypertensive patients
Authors: Harder Sebastian , Thürmann Petra A. , Ungethüm Wolfram
Published: November 2003

Aims
 To investigate the pharmacokinetic profile of the ACE‐inhibitor imidapril in 10 hypertensive patients after a first single dose (10 mg) and after 28 days therapy with imidapril 10 mg once daily.

Methods
 Cmax, tmax, t1/2 and AUC of imidapril and imidaprilat were obtained. ACE‐activity and arterial blood pressure during imidapril were corrected by a preceding placebo‐investigation.

Results
 The AUC of imidapril was 140 (43 s.d.) ng ml−1 h after the first dose and 123 (34 s.d.) ng ml−1 h at steady state. AUC of the active moiety imidaprilat averaged 211 (101 s.d.) ng ml−1 h after the first dose and 240 (55 s.d.) ng ml−1 h at the steady state investigation. Maximal ACE‐inhibition was 75% after the single dose as well as at steady state. ACE inhibition before drug intake at day 28 (i.e. trough) was 50%. The (placebo‐corrected) maximal drop in diastolic blood pressure after imidapril was 22 mmHg after the first dose and 25 mmHg at steady state. Exploratory analysis of imidaprilat plasma concentration vs effect profiles suggests a hyperbolic concentration effect relationship where data of the single dose contribute to the ascending part of an Emax‐curve, whereas the plateau around Emax is maintained at steady state.

Conclusions
 In this group of hypertensive patients, the pharmacokinetic profile and the drop in ACE‐activity as well as in blood pressure seen after a single dose of imidapril and at steady state were similar. The initial response to a test dose might therefore predict the response during chronic dosing.
Concentrations and effects of buspirone are considerably reduced by rifampicin
Authors: Lamberg T. S. , Kivistö K. T. , Neuvonen P. J.
Published: November 2003

Aims
 The effects of rifampicin on the pharmacokinetics and pharmacodynamics of buspirone, a non‐benzodiazepine anxiolytic agent, were investigated.

Methods
 In a randomized, placebo‐controlled cross‐over study with two phases, 10 young healthy volunteers took either 600 mg rifampicin or matched placebo once daily for 5 days. On day 6, 30 mg buspirone was administered orally. Plasma buspirone concentrations and effects of buspirone were measured up to 10 h.

Results
 The total area under the plasma buspirone concentration‐time curve after rifampicin was 10.4% (95% CI, 6.3–14.5%) of that after placebo (1.64±0.35 ng ml−1 h vs 22.0±15.1 ng ml−1 h (mean±s.d.); P<0.01). Rifampicin decreased the peak plasma concentration of buspirone from 6.6±3.7 ng ml−1 to 0.84±0.23 ng ml−1 (P<0.01) and the half‐life from 2.8±0.7 h to 1.3±0.5 h (P<0.01). A significant (P<0.05) reduction in the effects of buspirone was observed in three of the six psychomotor tests employed (postural sway test with eyes closed, subjective drowsiness and overall drug effect) after rifampicin pretreatment.

Conclusions
 The strong interaction between rifampicin and buspirone is probably mostly due to enhanced CYP3A4‐mediated first‐pass metabolism of buspirone. Buspirone will most likely show a greatly reduced anxiolytic effect when used together with rifampicin or other potent inducers of CYP3A4 such as phenytoin and carbamazepine.
Multifocal motor neuropathy caused by a B‐cell lymphoma producing a monoclonal IgM autoantibody against peripheral nerve myelin glycolipids GM1 and GD1b
Authors: Noguchi Masaaki , Mori Kiyoshi , Yamazaki Shigetaka , Suda Kouichi , Sato Naotake , Oshimi Kazuo
Published: November 2003

Summary.  Various data support the pathogenetic significance of serum IgM autoantibodies against glycolipid GM1 in patients with multifocal motor neuropathy. Although some patients with this neuropathy have an extraneural lymphoma, IgM anti‐GM1 glycolipid autoantibodies have not been investigated in these cases. We found IgM anti‐GM1 autoantibody in the serum of a 52‐year‐old man who developed multifocal motor neuropathy that was associated with an extraneural diffuse large B‐cell lymphoma. An autopsy showed severe widespread demyelination without lymphoma cell infiltration in the peripheral nerves. Immunofluorescent flow cytometry and thin‐layer chromatographic immunostaining demonstrated that most of the anti‐GM1 antibody in the serum was monoclonal IgM of λ type, which was also demonstrable in secretory form on lymphoma cells. The antibody showed affinity for the Galβ1‐3GalNAc terminal disaccharide of glycolipids GM1 and GD1b, which both are widespread in peripheral nerve myelin. Enzyme‐linked immunosorbent assay demonstrated that this antibody was much more abundant in lymphoma cell culture supernatant than in normal lymphocyte culture supernatant. Thus, our patient's B‐cell lymphoma cells produced a monoclonal IgM  λ autoantibody against this terminal disaccharide residue. This antibody bound to glycolipids GM1 and GD1b in peripheral motor nerve myelin, presumably initiating formation of destructive immune complexes that caused multifocal motor neuropathy.
Smoldering multiple myeloma: natural history and recognition of an evolving type
Authors: Rosiñol L. , Bladé J. , Esteve J. , Aymerich M. , Rozman M. , Montoto S. , Giné E. , Nadal E. , Filella X. , Queralt R. , Carrió A. , Montserrat E.
Published: November 2003

Summary.  Patients with smoldering multiple myeloma (SMM) meet the diagnostic criteria of multiple myeloma (MM) but are asymptomatic. Between January 1978 and July 2001, 53 patients (median age 63 years) were diagnosed with SMM. The median serum M‐protein and proportion of bone marrow plasma cells were 36 g/l and 27% respectively. Two subsets of SMM were identified: (i) evolving SMM (n = 22), characterized by a progressive increase in serum M‐protein, a previously recognized monoclonal gammopathy of undetermined significance (MGUS) and a significant higher proportion of IgA type and (ii) non‐evolving SMM (n = 26) with stable M‐protein that abruptly increases when symptomatic MM develops. Thirty‐four patients developed symptomatic MM. The median time to progression in the overall series was 3·2 years and the only feature associated with a shorter time to progression was the evolving versus non‐evolving type (1·3 vs. 3·9 years respectively, P = 0·007). The pattern of progression consisted of anaemia, lytic bone lesions or both, without renal failure, hypercalcaemia or extramedullary plasmacytomas. Fifty‐seven per cent of patients that required chemotherapy showed no or minimal response. The median survival from diagnosis and from progression was 8·2 and 3·5 years respectively.
Epigenetic alteration of the SOCS1 gene in chronic myeloid leukaemia
Authors: Liu Ta‐Chih , Lin Sheng‐Fung , Chang Jan‐Gowth , Yang Ming‐Yu , Hung Shih‐Ya , Chang Chao‐Sung
Published: November 2003

Summary.  The expression of the suppressor of cytokine signalling‐1 (SOCS1) protein is induced in response to stimulation by several cytokines. The induced SOCS1 inhibits the signalling pathway through the association with a variety of tyrosine kinase proteins. In this study, the mutation analyses, CpG island methylation status, and the expression of the SOCS1 gene in 112 chronic myeloid leukaemia (CML) samples, five leukaemia cell lines, and 30 normal controls were analysed. No genetic mutations of SOCS1 gene were noted in the CML samples. The SOCS1 gene was hypermethylated in 67% and 46% of the blastic and chronic phase CML samples respectively (P < 0·0001). However, there was no methylation of the SOCS1 gene in normal controls or CML in molecular remission. The methylation status of the SOCS1 gene is consistent with the results of the real‐time quantitative reverse transcription polymerase chain reaction and immunocytochemistry staining. Our results demonstrate that the SOCS1 gene silencing is caused by the methylation of CpG islands in CML and is reversed to an unmethylated status in molecular remission. As SOCS1 has universal activity to negatively regulate several cytokine signalling pathways, the loss of the negative regulation of cytokine signalling by the SOCS1 may play a role in the pathogenesis of CML progression.
Alterations of RAS signalling in Chinese multiple myeloma patients: absent BRAF and rare RAS mutations, but frequent inactivation of RASSF1A by transcriptional silencing or expression of a non‐functional variant transcript
Authors: Ng Margaret H. L. , Lau K. M. , Wong W. S. , To K. W. , Cheng S. H. , Tsang K. S. , Chan Natalie P. H. , Kho Bonnie C. S. , Lo K. W. , Tong Joanna H. M. , Lam C. W. , Chan Joyce C. W.
Published: November 2003

Summary.  The methylation status, mutation and expression of RASSF1A, and mutations of RAS and BRAF were studied in 52 patients with multiple myeloma (MM), one plasma cell leukaemia (PCL) patient and four MM‐derived cell lines. Aberrant methylation of RASSF1A was found in nine of 32 MM patients and in one of four MM cell lines (U266), where the associated loss of transcription was reversible by demethylation treatment. RASSF1A transcription was further investigated on anti‐CD138‐sorted plasma cell‐enriched bone marrow samples from 10 MM, one PCL and three reactive plasmacytosis patients. While the wild‐type RASSF1A transcript was detected in all three reactive plasmacytosis and the PCL samples, we found no detectable wild‐type transcripts in six of 10 MM samples studied. In two MM samples, only the non‐functional variant transcript was detected, whereas the other four showed loss of transcription. In great contrast to western data, RAS mutations were identified in only four of 31 (13%) MM patients. While no RASSF1A or BRAF mutation (V599E) was detected in any of the primary MM studied (n = 21), the latter was found in the U266 cell line. Taken together, these data indicate that alterations of RAS signalling are critical in MM pathogenesis. In our current studies of Chinese MM patients, these alterations involved frequent RASSF1A inactivation (60%) as a result of transcriptional silencing or expression of a non‐functional variant transcript.
Murine marrow‐derived mesenchymal stem cell: isolation, in vitro expansion, and characterization
Authors: Meirelles Lindolfo da Silva , Nardi Nance Beyer
Published: November 2003

Summary.  In spite of the attention given to the study of mesenchymal stem cells (MSCs) derived from the bone marrow (BM) of humans and other species, there is a lack of information about murine MSCs. We describe the establishment of conditions for the in vitro expansion of plastic‐adherent cells from murine BM for over 50 passages, and provide their characterization regarding morphology, surface marker profile and growth kinetics. These cells were shown to differentiate along osteogenic and adipogenic pathways, and to support the growth and differentiation of haematopoietic stem cells, and were thus operationally defined as murine mesenchymal stem cells (mMSCs). mMSCs were positive for the surface markers CD44, CD49e, CD29 and Sca‐1, and exhibited a homogeneous, distinctive morphology. Their frequency in the BM of adult BALB/c and C57Bl/6 mice, normal or knockout for the α‐L‐iduronidase (IDUA) gene, was preliminarily estimated to be 1 per 11 300–27 000 nucleated cells. The emergence of a defined methodology for the culture of mMSCs, as well as a comprehensive understanding of their biology, will make the development of cellular and genetic therapy protocols in murine models possible, and provide new perspectives in the field of adult stem cells research.
Impact of unrelated donor status on the incidence and outcome of cytomegalovirus infections after non‐myeloablative allogeneic stem cell transplantation
Authors: Junghanss Christian , Storb Rainer , Maris Michael B. , Carter Rachel A. , Sandmaier Brenda M. , Maloney David G. , McSweeney Peter A. , Corey Lawrence , Boeckh Michael
Published: November 2003

Summary.  Little is known about the impact of cytomegalovirus (CMV) infections that occur after human leucocyte antigen (HLA)‐matched unrelated donor (MUD) non‐myleoablative haematopoietic stem cell transplantation (HCT). We analysed the incidence, onset and outcomes of CMV infections in 59 recipients of MUD and in 109 recipients of HLA‐matched related donor (MRD) allogeneic HCT following non‐myeloablative conditioning containing 2 Gy total body irradiation and fludarabine. In CMV seropositive recipients, antigenaemia occurred in 68% (MUD) and in 49% (MRD, P = 0·08); there were no differences in the maximum levels of CMV antigenaemia and the time to cessation with antiviral therapy. CMV viraemia by culture was more common in MUD compared with MRD HCT recipients in univariate analysis (26% vs. 6%, P = 0·01), however, this difference was not detectable after controlling for other factors. The rates of CMV disease in the first 100 d were similar in MUD (9%) and MRD (5%) HCT recipients. CMV disease tended to occur earlier in the MUD compared with the MRD recipients (median day 41 vs. day 80). Beyond day 100, rates of CMV disease remained similar in both cohorts (cumulative incidence: MUD 21% and MRD 14%). The 30‐d and 1‐year survivals after CMV disease diagnosis were not significantly different in both groups. Thus, there appeared to be a trend toward increased CMV reactivation in MUD compared with MRD non‐myeloablative allogeneic HCT recipients; however, these differences did not reach statistical significance in this cohort and preemptive therapy was similarly effective in preventing CMV diseases.
The impact of retroviral suicide gene transduction procedures on T cells
Authors: Qasim Waseem , King Douglas , Buddle Jo , Verfuerth Stephanie , Kinnon Christine , Thrasher Adrian J. , Gaspar Hubert B.
Published: November 2003

Summary.  Retroviral vectors encoding the herpes simplex thymidine kinase gene have been used to render T cells sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used in clinical trials for their graft‐versus‐leukaemia effects following allogeneic haematopoietic stem cell transplantation. In the event of graft‐versus‐host disease (GVHD) the cells were susceptible to elimination through exposure to ganciclovir. We have investigated the impact of T‐cell activation, required for successful retrovirus‐mediated gene transfer, on T‐cell receptor repertoire profile, subset distribution and antiviral potential. Using a combination of antibodies against CD3 and CD28, T cells were transduced at high efficiency when exposed to retrovirus between 48 and 72 h later. Lymphocytes had undergone up to seven cycles of cell division by the end of the procedure. Although the T‐cell receptor Vβ repertoire was not altered after retroviral transduction, there were notable shifts in subset profiles with an increased proportion of CD45RO cells in transduced populations. T cells continued to proliferate for several days after transduction and were difficult to sustain under the extended culture conditions required to generate virus‐specific T cells. These observations may explain the lower than expected levels of GVHD and poor antiviral immunity reported in recent trials.
Functional and kinetic characterization of granulocyte colony‐stimulating factor‐primed CD34− human stem cells
Authors: Lemoli Roberto M. , Bertolini Francesco , Petrucci Maria T. , Gregorj Chiara , Ricciardi Maria R. , Fogli Miram , Curti Antonio , Rabascio Cristina , Pandolfi Simona , Ferrari Sergio , Fo Robin , Baccarani Michele , Tafuri Agostino
Published: November 2003

Summary.  We assessed the functional properties and the kinetic status in vitro, and the engraftment potential in vivo of human haematopoietic stem cells according to the expression of CD34 antigen. Lin−CD34− and Lin−CD34+ cells were isolated from granulocyte colony‐stimulating factor‐primed peripheral blood (PB) cells of healthy donors. The CD34− cell fraction did not contain either clonogenic cells in semisolid culture or long‐term culture initiating cells (LTC‐IC). However, stroma‐dependent liquid cultures and cytokines induced CD34 expression on a minority of stem cells, acquisition of clonogenic capacity and generation of LTC‐IC. Significantly higher percentages of quiescent G0 cells and lower percentages of cycling G1 cells were found in Lin−CD34− cells when compared with Lin−CD34+ cells. Kinetic quiescence of Lin−CD34− cells was associated with a significantly higher expression of the negative regulators of the cell cycle, p27Kip1 and p21cip1/waf1. Cytokine‐mediated induction of CD34, in vitro, resulted in cycling of stem cells and downregulation of p27. There was a higher rate of human long‐term engraftment in immunocompromised non‐obese diabetic (NOD)/recombination activating gene 1null and NOD/severe combined immunodeficient‐β2microglobulinnull mice injected with CD34+ cells. Thus, our study indicated that CD34 expression on human PB stem cells was associated with haematopoietic activity, cell‐cycle recruitment and downregulation of p27Kip1in vitro and higher engraftment capacity in vivo.
The effects of vitamin E on platelet activity in β‐thalassaemia patients
Authors: Unchern Supeenun , Laoharuangpanya Narumol , Phumala Noppawan , Sipankapracha Pornpan , Pootrakul Pensri , Fucharoen Suthat , Wanachivanawin Wanchai , Chantharaksri Udom
Published: November 2003

Summary.  A double‐blind, crossover, placebo‐controlled study of the effect of vitamin E on platelet functions was performed on nine splenectomized and 16 non‐splenectomized β‐thalassaemia/haemoglobin E (β‐thalassaemia/HbE) patients. The patients were supplemented with a daily dose of vitamin E (525 IU) for 3 months. The functions of platelets were assessed by adenosine diphosphate (ADP)‐induced platelet aggregation and adenosine triphosphate release. Plasma α‐tocopherol, plasma thiobarbituric reactive substances (TBARs) and serum ferritin levels represented patients’ antioxidant status, lipid peroxidation status and iron status respectively. Before experimentation, all patients had low plasma α‐tocopherol levels. The splenectomized patients showed severe iron overload iron, had higher plasma TBAR levels and their platelets were more reactive to ADP than those of non‐splenectomized patients. Three months of daily vitamin E supplementation resulted in a significant increase in plasma α‐tocopherol levels and reduction in plasma TBAR levels in all patients. Serum ferritin levels of the patients were not altered; however, vitamin E reduced the platelet reactivity of the splenectomized patients towards normal levels. The influence of vitamin E on platelet reactivity may result in delaying hypoxaemia and pulmonary occlusion that commonly occurs in splenectomized β‐thalassaemia/HbE patients.
Proliferative response of mouse spermatogonial stem cells after irradiation: a quantitative model analysis of experimental data
Authors: Judas L. , Bentzen S. M. , Hansen P. V. , Overgaard J.
Published: November 2003

The testes of CDF1 mice were irradiated with single doses of X‐rays ranging from 2–16 Gy. The number of haploid cells in the testis at different times after irradiation (42–350 days) was determined by one‐parameter flow cytometry both for irradiated animals and for age‐matched controls. Based on literature data on the kinetics of the spermatogenesis in mice, a mathematical model of the (hierarchical) germ tissue was developed. Using this model, the processes of radiation‐induced cell loss and subsequent recovery were simulated and free parameters of the model were estimated by fitting the model prediction to the experimental data. One of the aims of the study was to investigate the kinetic behaviour of spermatogonial stem cells and the corresponding control mechanisms.

In order to fit the data, the model has to include the following features: (i) A preferential self‐repopulation of spermatogonial stem cells following tissue injury. The model‐estimated probability of a self‐renewing division rises from 50% (the steady‐state value) to 95% if the stem‐cell population is reduced to 10% of its normal size. (ii) A relatively low, almost constant turnover rate of the stem‐cell compartment. It is suggested by the analysis that less than 10% of the permatogonial stem cells present in the testis divide per day, regardless of the degree of cellular depletion. (iii) A mechanism responsible for incomplete recovery. The observed incomplete recovery of spermatogenesis after single doses exceeding 10 Gy can be described quantitatively assuming that the stem cells are organized into discrete proliferative structures, the number of cells per structure being about 60.
Genesis and evolution of high‐ploidy tumour cells evaluated by means of the proliferation markers p34cdc2, cyclin B1, PCNA and 3[H]‐thymidine
Authors: Baroja A. , De La Hoz C. , Alvarez A. , Ispizua A. , Bilbao J. , De Gandarias J. M.
Published: November 2003

Although cell polyploidization is not an infrequent event in mammalian cells and is common in tumours, the mechanisms involved are not well understood. Using the murine B16 cell line as a model, we evaluated the role of some key proteins involved in cell cycle progression: p34cdc2, cyclin B1 and PCNA. By means of flow cytometry, we showed that both in modal‐ and in high‐ploidy subpopulations, almost all cells were p34cdc2‐positive. In the modal‐ploidy subpopulation only 17.1% cells were cyclin B1‐positive and 85.6% PCNA‐positive; in contrast, in the high‐ploidy subpopulation up to 91.8% cells were cyclin B1‐positive and 97.3% cells were PCNA‐positive (P < 0.001). Immunofluorescence microscopy showed that PCNA was located in the nucleus; p34cdc2, both in the nucleus and cytoplasm; and cyclin B1 yielded a cytoplasmic spotted pattern with a perinuclear reinforcement. After a 24‐h incubation with 3[H]‐thymidine followed by withdrawal of the isotope, high‐ploidy cells remained labelled 8 days after thymidine withdrawal, in contrast to modalploidy cells. Taken together, our results suggest that polyploid cells are not quiescent, their cell cycle is longer than that of the modal‐ploidy population, and they maintain cyclin B1 throughout the cycle, which may contribute to their genesis by impeding the exit from mitosis.
Circadian variation of DNA replication in hamster cheek pouch epithelium analysed by tritiated thymidine labelling, flow sorting and autoradiography: no resting S phase cells in the normal epithelium
Authors: Larsen J. K. , Møller U. , Christensen I. J. , Christiansen J.
Published: November 2003

In the normal hamster cheek pouch epithelium, cell proliferation takes place with a pronounced circadian rhythm. We tested our previous hypothesis that all cells having S phase DNA content are actively synthesizing DNA and thus participating in the daily cohort of proliferating cells. We found no evidence of resting S phase cells in the normal epithelium. Using labelling with tritiated thymidine followed by fluorescence activated cell sorting according to DNA content and by autoradiography of the sorted nuclei, it was demonstrated that during the 24 h period almost all cells with mid S phase DNA content were active in DNA synthesis.
Isolation and culture of hamster ectoplacental cone trophoblasts: an in vitro study on the cell types and their growth pattern
Authors: Pande S. , Shukla R. , Mehrotra P. K. , Kamboj V. P.
Published: November 2003

The method of isolation of trophoblast cell types from ectoplacental cone of hamster embryo of day 8 post coitum (plug day as day 1) and examination of their growth pattern in vitro is presented in this study. Based on their size, three types of trophoblast cells were identified: (1) the smaller cells having clear cytoplasm formed the major portion (70% to 80%) of the total cell population (2) moderately bigger cells having mono or binucleated appearance and containing minute granules in the nuclear region formed 5% to 10% and (3) extra large and multinucleated cells shared 10% to 20% of the total cell number. While the smaller and slightly bigger cells showed moderate growth the larger ones had extensive growth and were seen to acquire different shapes on extending the duration of culture, such as fusiform, dumb‐bell, polygonal, rectangular or flowery. The extremities of the cytoplasmic growth of these cells were seen to be thickened at one end giving the impression of a pad‐like structure. The significance of these adaptations are not known at present.
Effects of calcitonin gene‐related peptide and interleukin 6 on myoblast differentiation
Authors: Okazaki S. , Kawai H. , Arii Y. , Yamaguchi H. , Saito S.
Published: November 2003

Effects of calcitonin gene‐related peptide (CGRP) and interleukin 6 (IL‐6) on muscle cell differentiation were studied using cultured rat myoblasts (L6 cells). Cell morphology and the amounts of the messenger RNAs (mRNAs) of myogenin and Myf‐5, DNA content, creatine kinase (CK) activity, and myoglobin (Mb) content in the cultured cells were examined serially over 10 days of culture. In the presence of CGRP or IL‐6, the mRNAs of myogenin and Myf‐5 were expressed earlier and at a higher concentration in the treated cells than in the control cells. The ratios of CK activity to DNA content (CK/DNA) and of Mb content to DNA content (Mb/DNA) on day 10 of culture also were greater than in the control cells. Furthermore, the mRNAs of myogenin and Myf‐5 in cultured cells incubated with both CGRP and IL‐6 increased more rapidly than in cells cultured with CGRP or IL‐6 alone, and the ratios of CK/DNA and Mb/DNA on day 10 were more than twice those in the presence of CGRP or IL‐6. These findings indicate that both CGRP and IL‐6 facilitate the differentiation of myoblasts and may have an additive effect.
Expression of proliferation‐associated nuclear autoantigens, p330d/CENP‐F and PCNA, in differentiation and in drug‐induced growth inhibition using two‐parameter flow cytometry
Authors: Báez A. , Torres K. , Tan E. M. , Pommier Y. , Casiano C. A.
Published: November 2003

p330d/CENP‐F is a recently described nuclear autoantigen that was detected in PHA‐stimulated but not in resting peripheral lymphocytes. This protein accumulates in the nucleus during S‐phase and reaches maximum levels during the G2 and M phases of the cell cycles. We compared the expression of p330d/CENP‐F and proliferating cell nuclear antigen (PCNA) during the induction of terminal myeloid differentiation of HL‐60 tumour cells. HL‐60 cells were induced to differentiate with retinoic acid (RA), dimethyl sulfoxide (DMSO), and 3‐nitrobenzothiazolo [3,2‐]quinolinium (NBQ), and collected at different intervals. Control and treated cells were analyzed by two‐parameter flow cytometry using propidium iodide and antibodies to p330d/CENP‐F and PCNA. The percentage of p330d/CENP‐F and PCNA positive cells was found to be proportional to the percentage of proliferating cells. After two cell cycles (65 h), the percentage of p330d/CENP‐F and PCNA positive cells was reduced proportionately to the number of cells that had differentiated. Reduction in the expression of both antigens was completed after 120 h when 80% to 85% of the cells were arrested in G1 and displayed the mature phenotype. The expression of p330d/CENP‐F and PCNA was also assessed in the growth inhibition of HT‐29 cells induced by various concentrations of camptothecin (CPT), etoposide (VP‐16), and aphidicolin (APH). There was a dose‐dependent displacement of cells to late S‐phase by CPT while VP‐16 induced cells to accumulate in G2+ M, and as expected these effects caused a strong increase in the cellular levels of both antigens. The arrest of cells in G1 by APH led to a significant decrease in their expression. The dramatic reduction in p330d/CENP‐F levels during differentiation, and the correlation of its expression with the cell cycle effects of the cytotoxic drugs are consistent with the behaviour expected for a proliferation marker.
The relative spatial distribution of in vitro‐CFCs in the bone marrow, responding to specific growth factors
Authors: Cui Y.‐F. , Lord B. I. , Woolford L. B. , Testa N. G.
Published: November 2003

Haemopoietic progenitor cells are stimulated by a range of growth factors which promote colony growth in culture. The progenitors are a part of an age‐structured developmental hierarchy in the tissue. The growth factors, although overlapping in their effects, stimulate cells preferentially at different stages in this programme. Femoral bone marrow was fractionated into axial (close to the central venous sinus) and marginal (close to the bone surface) cells. Progenitors which responded to IL‐3, GM‐CSF, G‐CSF, M‐CSF and SCF were then assayed in soft agar cultures. Consequent plots of their spatial distributions showed that the more primitive cells in vitro (responding to IL‐3) were concentrated close to the bone surface. The peak concentrations of cells responding primarily to growth factors with progressively more affinity to more mature progenitor cells correspondingly appeared progressively further from the bone surface and closer to the point of release at the central venous sinus. This suggests that the developmental/maturational process in haemopoiesis is accompanied by a progressive movement of cells from the bone surface towards the central axial regions of the bone cavities. The most primitive cells are however exposed, close to the centre of the cavity, by a combination of SCF and G‐CSF (or by a 50‐fold increase in G‐CSF concentration alone). These results corroborate earlier data which indicate a developmental movement of cells from the centre of the marrow tissue towards the bone surface and back again, sequentially encountering a series of growth factors which promote their differentiation into mature cells, for release at the central venous sinus.
Unscheduled expression of cyclins D1 and D3 in human tumour cell lines
Authors: Juan G. , Gong J. , Traganos F. , Darzynkiewicz Z.
Published: November 2003

D‐type cyclins are involved in regulation of cell traverse through G1 primarily by activating the cyclin‐dependent kinase 4 (CDK4) and targeting it to the retinoblastoma tumour suppressor protein. There is a vast body of evidence that defective expression of D‐type cyclins is associated with tumour development and/or progression. Immunocytochemical detection of D cyclins combined with multiparameter flow cytometry makes it possible to measure the expression of these proteins in individual cells in relation to their cell cycle position without the need for cell synchronization. This approach was used in the present study to compare the cell cycle phase specific expression of cyclins D3 and D1 in human normal proliferating lymphocytes and fibroblasts, respectively, with nine tumour cell lines of different lineage. During exponential, unperturbed growth, expression of cyclin D1 in fibroblasts from donors of different age, or cyclin D3 in lymphocytes, was limited to mid‐G1 cells: Less than 7% of the cells entering S phase or progressing through S and G2 were cyclin D positive. In contrast, expression of either cyclin D1 or cyclin D3 in tumour cell lines of different lineage was not limited to G1 phase. Namely, over 80% of the cells in S and G2+M were cyclin D positive in eight of the nine cell lines studied. The data indicate that while expression of cyclin D1 or D3 in normal cells is discontinuous, occurring transiently in G1, these proteins are expressed in some tumour lines persistently throughout the cell cycle. This suggests that the partner kinase CDK4 is perpetually active throughout the cell cycle in these tumour lines.
Quantitative Clinical Pathology
Authors: McMichael‐Phillips D.
Published: November 2003

Edited by Peter Hamilton and Derek Allen. 1995. Blackwell Science, Oxford. 342 pages. Hardback. ISBN 0‐632‐03286‐3.
p53 mutations associated with increased sensitivity to ionizing radiation in human head and neck cancer cell lines
Authors: Servomaa K. , Kiuru A. , Grénman R. , Pekkola‐Heino K. , Pulkkinen J. O. , Rytömaa T.
Published: November 2003

The p53 tumour suppressor gene is activated following cellular exposure to DNA‐damaging agents. The functions of wild‐type p53 protein include transient blocking of cell cycle progression, direct or indirect stimulation of DNA repair machinery and triggering of apoptosis if DNA repair fails. Therefore, the status of p53 protein may be critically associated with tumour cell radiosensitivity.

In the present study we examine the intrinsic radiosensitivity of 20 human carcinoma cell lines derived from 15 patients with different types of head and neck tumour. Radiosensitivities were measured in a 96‐well plate clonogenic assay in terms of the mean inactivation dose, surviving fraction at 2 Gy, and constants α and β in the linear quadratic survival curve. The p53 allele status was determined by amplifying exons 4–10 by the polymerase chain reaction (PCR), screening for mutations using single‐strand conformation polymorphism (SSCP) analysis and determining the exact type and location of a mutation by direct sequencing. The results showed that prevalence of p53 mutations in squamous cell carcinoma (SCC) cell lines is high (80%), and that deletion of one or both wild‐type alleles is common (75%). Intrinsic radiosensitivity of the cell lines varied greatly in terms of mean inactivation dose, from 1.4±0.1 to 2.6±0.2 Gy. Radiosensitivity correlated well with the p53 allele status so that cell lines carrying a wild‐type p53 allele were significantly (P<0.01) more radioresistant (mean inactivation dose 2.23±0.15 Gy) than cell lines which lacked a wild‐type gene (1.82±0.24 Gy).

Evaluation of our own results and those published in the literature lead us to conclude that absence of the wild‐type p53 allele in human head and neck cancer cell lines is associated with increased radiosensitivity. However, the sensitivity is also strongly dependent on the exact type and location of the p53 mutation.
Molecular characterization of the gene locus of the human cell proliferation‐associated nuclear protein defined by monoclonal antibody Ki‐67
Authors: Duchrow M. , Schlüter C. , Wohlenberg C. , Flad H‐D. , Gerdes J.
Published: November 2003

The human antigen defined by the monoclonal antibody Ki‐67, the ‘Ki‐67 protein’, is an ubiquitously expressed human nuclear protein strictly associated with cell proliferation and is widely used in routine pathology as a ‘proliferation marker’ to measure the growth fraction in human tumours. In immunoblots of proteins from proliferating cells, Ki‐67 detects two bands with the apparent molecular weights of 345 and 395 kDa. Recently we reported on the cloning and sequencing of the complete cDNA of the Ki‐67 protein. We found two isoforms of cDNA with full lengths of 11.4 and 12.5 kb, respectively, likely formed by the alternative splicing of exon 7. The remarkable exon 13 at the ‘centre’ of this gene contains 16 homologous segments of 366 bp (Ki‐67 repeats), each including a highly conserved new motif of 66 bp (Ki‐67 motif). Computer analyses confirmed that the cDNAs encode for a new class of nuclear proteins.

The complete gene locus of the Ki‐67 protein, comprising a 74 bp 5′ region and a 264 bp 3′ region, has been sequenced and aligned to a continuous sequence of 29 965 bp length located on chromosome 10q25‐ter. The gene is organized in 15 exons with sizes from 67 to 6845 bp and in 14 introns with sizes from 87 to 3569 bp. Three introns contain homologue copies of ‘Alu‐repeats’. Interestingly, the introns flanking the alternative spliced exon 7 are free of any consensus donor and acceptor splicing signal. All other intron‐exon transitions contained a potential branch site. The complete 5′ region including the first two exons represents a CpG‐rich island. We found the transcription initiation site in exon two adjacent to the consensus sequence of a cap site. Upstream of this cap site no TATA‐ or CCAAT‐box could be located, but downstream we found two remarkable directly repeated elements of 24 bp lengths each containing a TATA box in inverse orientation.