The in vitro anti-denaturation effects induced by natural products and non-steroidal compounds in heat treated (immunogenic) bovine serum albumin is proposed as a screening assay for the detection of anti-inflammatory compounds, without the use of animals, in the early stages of the drug discovery process.

There are emerging ethical issues with regards to the use of animals in the early stages of drug discovery for anti-inflammatory and degenerative diseases from natural products using the activity-directed isolation pathways when many compounds (eg > 100) are present in the crude extract or fraction and are to be tested The above-mentioned is the main reason for proposing the use of the in vitro anti-denaturation (stabilization) effects of heat treated (immunogenic) bovine serum albumin (BSA) as an assay. Current methods used for detecting and isolating a wide range of anti-inflammatory compounds in the early stages of the drug discovery process utilize a large number of animals. When BSA is heated and is undergoing denaturation, it expresses antigens associated to Type III hypersensitive reaction and which are related to diseases such as serum sickness, glomerulonephritis, rheumatoid arthritis and systemic lupus erythematosus. Thus, the assay that is being proposed should be applicable to the discovery of drugs for treating the above mentioned diseases and others, once the compounds stabilize the denaturation process.

Immunological evidence supporting the use of extracts from Boehmeria jamaicensis Urb for treating the common cold and sinus infections

Mixed lymphocyte responses assays were conducted at 25.0 and 250.0 microg/mL of the crude ethanolic extract of Boehmeria jamaicensis Urb (coded as BJE) using peripheral lymphocytes obtained from individuals suffering from the common cold after four days of infection and from healthy individuals (without the common cold infection). At a concentration of 25 ug/mL, gamma interferon (IFN-gamma) was increased by 24.03 fold and interleukin 4 (IL-4) by 1.71 fold for the cells obtained from individuals with the common cold (Group A). The extract suppressed IFN-gamma by 8.3% while IL-4 was stimulated by 9.90 fold from peripheral lymphocytes obtained from healthy individuals (Group B). Gamma interferon was suppressed at 250 microg/mL while IL-4 was elevated by 1.86 fold for cells obtained from individuals suffering from the common cold (Group A). In conclusion, BJE could have implications for the treatment of the common cold.

Ensayos de reacci¨®n linfocitaria mixta fueron realizados a 25.0 y 250.0 ¦Ìg/mL de extracto etan¨®lico crudo de Boehmeria jamaicensis Urb (codificado como BJE), usando linfocitos perif¨¦ricos obtenidos de individuos con catarro com¨²n luego de cuatro d¨ªas de infecci¨®n, y de individuos sanos (sin la infecci¨®n del catarro com¨²n). Se hall¨® que el interfer¨®n-gamma (IFN-¦Ã) aument¨® en 24.03 veces, y la interleucina 4 (IL-4) en 1.71 veces para las c¨¦lulas obtenidas de individuos con catarro com¨²n, a 25¦Ìg/mL. El extracto inhibi¨® IFN-¦Ã en un 8.3 % en tanto que el IL-4 fue estimulado en 9.90 veces a partirde los linfocitos perif¨¦ricos obtenidos de individuos sanos. El gamma-interfer¨®n fue inhibido a 250 ¦Ìg/mL, mientras que la IL-4 se elev¨® en 1.86 veces para las c¨¦lulas obtenidas de individuos que sufren de catarro com¨²n.

Immunological evidence supporting the use of extracts from Boehmeria jamaicensis Urb for treating the common cold and sinus infections.

Mixed lymphocyte responses assays were conducted at 25.0 and 250.0 microg/mL of the crude ethanolic extract of Boehmeria jamaicensis Urb (coded as BJE) using peripheral lymphocytes obtained from individuals suffering from the common cold after four days of infection and from healthy individuals (without the common cold infection). At a concentration of 25 ug/mL, gamma interferon (IFN-gamma) was increased by 24.03 fold and interleukin 4 (IL-4) by 1.71 fold for the cells obtained from individuals with the common cold (Group A). The extract suppressed IFN-gamma by 8.3% while IL-4 was stimulated by 9.90 fold from peripheral lymphocytes obtained from healthy individuals (Group B). Gamma interferon was suppressed at 250 microg/mL while IL-4 was elevated by 1.86 fold for cells obtained from individuals suffering from the common cold (Group A). In conclusion, BJE could have implications for the treatment of the common cold.

Low-threshold L-type calcium channels in rat dopamine neurons.

Ca(2+) channel subtypes expressed by dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) were studied using whole cell patch-clamp recordings and blockers selective for different channel types (L, N, and P/Q). Nimodipine (Nim, 2 microM), omega-conotoxin GVIA (Ctx, 1 microM), or omega-agatoxin IVA (Atx, 50 nM) blocked 27, 36, and 37% of peak whole cell Ca(2+) channel current, respectively, indicating the presence of L-, N-, and P-type channels. Nim blocked approximately twice as much Ca(2+) channel current near activation threshold compared with Ctx or Atx, suggesting that small depolarizations preferentially opened L-type versus N- or P-type Ca(2+) channels. N- and L-channels in DA neurons opened over a significantly more negative voltage range than those in rat dorsal root ganglion cells, recorded from using identical conditions. These data provide an explanation as to why Ca(2+)-dependent spontaneous oscillatory potentials and rhythmic firing in DA neurons are blocked by L-channel but not N-channel antagonists and suggest that pharmacologically similar Ca(2+) channels may exhibit different thresholds for activation in different types of neurons.

Muscarine reduces calcium-dependent electrical activity in substantia nigra dopaminergic neurons.

The effect of muscarine on Ca2+ dependent electrical activity was studied in dopamine (DA) neurons located in the substantia nigra pars compacta (SNc) in brain slices from young rats, using sharp electrodes. In most DA neurons tested, muscarine (50 microM) reduced the amplitude of spontaneous oscillatory potentials and evoked Ca2+-dependent potentials recorded in the presence of TTX. Muscarine also reduced the amplitude of the slow afterhyperpolarization (sAHP) following action potentials in most DA neurons. These data suggest that muscarine reduces Ca2+ entry in SNc DA neurons. The reduction of the amplitude of the sAHP by muscarine in DA neurons may facilitate bursting initiated by glutamatergic input by increasing the frequency at which DA neurons can fire. The reduction of the sAHP via activation of muscarinic receptors in vivo may provide a mechanism whereby cholinergic inputs to DA neurons from the tegmental peduncular pontine nucleus could modulate dopamine release at dopaminergic targets in the brain.

Characterization of prohibitin in a newly established rat ovarian granulosa cell line.

Prohibitin is an evolutionary conserved protein that is associated with cellular differentiation, atresia, and luteolysis in the rat ovary. However, the specific cellular location and function of prohibitin in ovarian cells has not been clearly elucidated. To characterize the expression of prohibitin during cell proliferation, differentiation, and cell death, we have successfully established a temperature-sensitive granulosa cell line, designated RGA-1. At a permissive temperature of 33 C, RGA-1 cells proliferate, but revert to a differentiated phenotype at a nonpermissive temperature of 39 C. Significant inductions of prohibitin mRNA and protein expression were observed in the differentiated phenotype when compared with proliferating cells. Differentiated RGA-1 cells were found to express inhibin alpha- and beta-transcripts, as well as steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor proteins in a manner reminiscent of steroidogenic functional responses observed in primary differentiated granulosa cells. Prohibitin expression correlated well with the expression of these steroidogenic proteins. At 39 C, RGA-1 cells also displayed increases in p53 protein levels, indicative of growth arrest in the nonproliferating cells. Confocal and electron microscopic examinations revealed increased prohibitin localization to the mitochondria at 39 C, along with changes in mitochondrial size and shape. These changes were accompanied by marked reductions in cytochrome c oxidase subunit II levels and in unit mitochondrial transmembrane potential. In addition, cell fractionation studies demonstrated that the prohibitin protein was mainly localized to the mitochondrial membrane. Collectively, these findings suggest a role for prohibitin in mitochondrial structure and function during growth and differentiation in ovarian granulosa cells. Prohibitin expression may also be indicative of mitochondrial destabilization during apoptosis-related events.

Long-term follow-up confirms the benefit of all-trans retinoic acid in acute promyelocytic leukemia. European APL group.

First results of a randomized trial (APL91 trial) and other randomized or non-randomized studies have shown that ATRA followed by chemotherapy significantly increased event-free survival (EFS) and survival, and decreased the incidence of relapse by comparison to chemotherapy alone in newly diagnosed APL. We present here long-term follow-up of the APL91 trial. In this trial, 101 patients had been randomized between ATRA followed by three courses of daunorubicin-AraC chemotherapy (ATRA group) and the same chemotherapy alone (chemotherapy group). Results were reanalyzed 73 months after closing of patient entry. Updated results of APL 91 trial found a Kaplan-Meier estimate of EFS and relapse rate at 4 years of 63% and 31% in the ATRA group, as compared to 17% and 78% in the chemotherapy group (P= 10(-4) and relative risk 2.95, P= 10(-4) and relative risk 3.68, respectively). Kaplan-Meier survival at 4 years was 76% in the ATRA group and 49% in the chemotherapy group (P= 0.026, relative risk 2.7). In the chemotherapy group, seven of the 27 relapses occurred after 18 months, but no relapse was seen after 43 months. In the ATRA group, four of the 17 relapses occurred after 18 months, including two late relapses (at 58 and 74 months). In the chemotherapy group, 23 of the 25 patients who relapsed achieved a second CR with ATRA, and the Kaplan-Meier estimate of second relapse was 40% at 30 months. In the ATRA group, the 10 patients who relapsed and were retreated with ATRA achieved a second CR. In conclusion, long-term results of APL91 trial confirm the superiority of the combination of ATRA and chemotherapy over chemotherapy alone in newly diagnosed APL, and that ATRA should be incorporated in the front-line treatment of APL.

Haematologists' approaches to the management of adolescents and young adults with acute leukaemia.

Approaches to the management of adolescents and young adults with acute leukaemia were investigated by sending a questionnaire to hospitals identified as having diagnosed or treated patients aged 15-29 years. The responses demonstrated the types of hospital treating these patients, the haematologists' perceived practice for entry of patients to Medical Research Council (MRC) leukaemia trials and reasons for non-entry. Data were linked to MRC trials data to determine the proportion of patients aged 15-29 years at diagnosis in responding hospitals actually treated in MRC leukaemia trials in the 5 years preceding the questionnaire. Eighty-two per cent of haematologists stated that they entered patients 'always' or 'whenever possible' for acute myeloid leukaemia (AML) and 76% for acute lymphoblastic leukaemia (ALL), but actual entry rates from the study hospitals were 46% of 239 AML patients and 36% of 182 ALL patients. The reasons most commonly reported for not entering eligible patients to national leukaemia trials were clinician preference for one arm of an MRC trial, a regional study or non-trial protocol, and concern about workload and ethical approval.

Deregulated expression of promyelocytic leukemia zinc finger protein in B-cell chronic lymphocytic leukemias does not affect cyclin A expression.

INTRODUCTION: The promyelocytic leukemia zinc finger (PLZF) gene encodes a transcription factor expressed in myeloid, lymphoid and CD34(+) progenitor cells. Structurally related to BCL-6, which is involved in human lymphoma, PLZF may have a role in proliferation, differentiation and survival of hematopoietic cells, that could be mediated by transcriptional repression of the cyclin A gene. MATERIALS AND METHODS: Quantitative competitive reverse transcription-polymerase chain reaction was used to measure the levels of expression of PLZF and cyclin A in normal leukocyte subsets (including CD19(+) lymphocytes, n=21) and malignant B lymphocytes (including B-chronic lymphocytic leukemias [B-CLL], n=63). Results obtained with this method were confirmed by Western and Northern blot analysis. Transactivation assays were performed using an expression construct for PLZF and two cyclin A promoter luciferase reporters in an Epstein-Barr virus (EBV)-transformed B-cell line. Cyclin A expression, cell growth kinetics, and cell cycle were analysed in stable clones of the Burkitt lymphoma (BL) B-cell line DG75 with inducible expression of PLZF, generated using the tetracycline-regulated expression system. RESULTS: Expression of PLZF was 100-fold downregulated in 90% B-CLL (56/63) compared to normal B lymphocytes (P<0.001). B-CLL patients with the highest levels of PLZF had a poorer survival (P<0.013). In transactivation assays, PLZF inhibited the activity of the cyclin A reporters by 50%, demonstrating that PLZF can repress cyclin A expression in non-malignant B lymphocytes. However, in B-CLL patients, the level of cyclin A expression was found to be within the normal range. Altered PLZF function in B lymphoid malignancies was further corroborated in the PLZF-regulatable DG75 clones, where induction of PLZF expression did not significantly alter the levels of cyclin A expression, the cell growth kinetics, or the cell cycle phase distribution. CONCLUSION: The lower survival of patients with the highest levels of PLZF suggests that this protein may be a marker of progression in B-CLL. The absence of co-ordinated regulation of PLZF and cyclin A genes in B-CLL and in a malignant B-cell line may indicate a loss of cyclin A control by PLZF in B-CLL and other B-cell disorders. Deregulation of PLZF could thus play a role in B-cell malignancy.

H RAS mutations in haematologically normal individuals.

INTRODUCTION: Point mutations in N, K and H RAS have been found in adverse haematological malignancies. The background frequency of RAS mutations in the normal population has yet to be determined. Here we report the results of a screen for RAS mutations from normal individuals. MATERIALS AND METHODS: DNA from peripheral blood or bone marrow from 115 haematologically normal individuals was screened for point mutations in N, K and H RAS, at amino acid positions 12, 13 and 61. The screening was done using polymerase chain reaction and oligonucleotide hybridisation and candidate mutations were subsequently confirmed by cloning and sequencing. RESULTS AND CONCLUSION: Point mutations were identified in DNA from two of the 115 individuals. Both mutations resulted in an amino acid substitution at position 12 in H RAS. Both individuals with detectable H RAS mutations remain haematologically normal.

Immunolocalization and expression of prohibitin, a mitochondrial associated protein within the rat ovaries.

This study was designed to determine the cellular distribution and pattern of expression for the mitochondria-associated protein, prohibitin, during the transitional stages of follicular differentiation within the rat ovary. Immunohistochemical staining techniques were used on frozen sections to examine the localization of prohibitin to preantral, antral, preovulatory, and atretic follicles. Prohibitin localization was also determined in corpus luteum from adult rats, in addition to those from infant and juvenile ovaries, before and after gonadotropin stimulation. Western and Northern blotting techniques were used for qualitative and quantitative assessment of prohibitin expression levels within the ovary. Prohibitin was localized within granulosa cells of infant and juvenile ovaries in a relatively heterogeneous staining pattern. The oocyte also exhibited robust prohibitin expression at all stages of follicular development. In addition, strong prohibitin expression was evident in the corpus luteum as well as in follicles undergoing atresia. Additional data derived from studies involving a GnRH-agonist indicate that increases in prohibitin protein expression correlate with the initial events of apoptosis. Collectively, these results support a growth regulatory role for prohibitin within the rat ovary. Therefore, we propose that prohibitin may serve as an important regulator of granulosa cell fate during follicular development.

Immunolocalization and expression of the steroidogenic acute regulatory protein during the transitional stages of rat follicular differentiation.

This study was designed to determine the pattern of expression and cellular distribution of the steroidogenic acute regulatory protein (StAR) during the transitional stages of follicular differentiation in rat ovary. Using specific antisera against the StAR, immunohistochemistry, Western blotting, and immunoprecipitation analyses provide evidence confirming the localization and expression of StAR in granulosa cells (GCs) of juvenile rat ovaries before and after PMSG treatment. The results also show that StAR expression occurs in theca intersitial cells surrounding preantral, antral, and larger antral follicles in adult diestrous ovaries. Furthermore, we have demonstrated heterogenous StAR immunoreactivity in the granulosa cell layers and cells of the corpora lutea. A novel finding presented here is that, during ongoing growth and differentiation of the follicle, the immunoreactivity of StAR tends to shift from the GC of early antral follicles to the theca cell layers in the adult. The spatiotemporal changes or shifts in StAR expression and cellular localization also coincide with the appearance of more acidic isoforms of the 30-kD protein, as determined by two-dimensional gel electrophoresis. Although the functional implications of these observations remain unclear, the acute temporal changes in StAR expression and localization may not only reflect the dynamic steroidogenic capacity of follicular cells but may also support a possible role for FSH in the induction of follicular maturation.

Alterations of the retinoic acid receptor alpha (RAR alpha) gene in myeloid and lymphoid malignancies.

The retinoic acid receptor alpha (RAR alpha) protein plays a central role in myeloid differentiation, and chromosomal translocations disrupting the RAR alpha gene are implicated in the development of acute myeloid leukaemia (AML). To identify haemopoietic malignant disorders which may also be linked to RAR alpha abnormalities, Southern blot analysis was performed in DNA from 153 patients with haematological malignancies other than AML FAB type M3 using RAR alpha cDNA probes. Alterations of RAR alpha were detected in 1/42 myelodysplastic syndromes (MDS), 2/24 AML, 3/47 B-chronic lymphocytic leukaemias (B-CLL), 0/40 lymphomas and 0/60 normal individuals. These data strongly suggest that alterations of RAR alpha might predispose for myeloid and lymphoid disorders.

Patterns of care and survival for adolescents and young adults with acute leukaemia--a population-based study.

We report a population-based study of patterns of care and survival for people with acute leukaemia diagnosed at age 15-29 years during 1984-94 in regions of England and Wales covered by specialist leukaemia registries. There were 879 patients: 417 with acute lymphoblastic leukaemia (ALL) and 462 with acute myeloid leukaemia (AML). For ALL, actuarial survival rates were 43% at 5 years after diagnosis and 37% at 10 years. Survival improved significantly between 1984-88 and 1989-94 for those aged 15-19 at diagnosis. Patients entered in national clinical trials and those not entered had similar survival rates. Survival rates were similar at teaching and non-teaching hospitals and at hospitals treating different numbers of study patients per year. For AML, survival rates were 42% at 5 years after diagnosis and 39% at 10 years. Survival improved significantly between 1984-88 and 1989-94. Patients entered in the Medical Research Council AML10 trial had a higher survival rate than those who were in the earlier AML9 trial. Survival did not vary with category of hospital. We conclude that survival has improved for adolescents and young adults with acute leukaemia but that there is at present no evidence that centralized treatment results in a survival benefit for patients in this age group.

Parenteral glutamine protects hepatic function during bone marrow transplantation.

Hepatic veno-occlusive disease (VOD) of the liver is a common complication following high-dose cytotoxic therapy for bone marrow transplantation (BMT). The major pathological changes are seen in centrilobular (zone 3) hepatocytes and adjacent endothelium. Glutathione (GSH) becomes depleted following chemotherapy and experimental evidence suggests reduced levels predispose to centrilobular hepatocyte and endothelial cell injury. Animal studies have shown that glutamine infusions can maintain GSH levels and protect against free radical injury. We have prospectively studied the effect of glutamine supplementation during BMT. Thirty-four patients undergoing BMT were randomised to receive either glycl-L-glutamine (n = 18) or an isonitrogenous mixture of non-essential amino acids (n = 16). Glutamine was shown to significantly preserve protein C (days +4 and +7, P < 0.05) and albumin levels (days 0 and +4, P < 0.02). Markers of thrombin and plasmin generation (thrombin-antithrombin, prothrombin fragment F1+2 and plasmin-antiplasmin levels) were not significantly changed between the two groups. These findings suggest that glutamine preserves hepatic function but does not alter thrombin or plasmin generation during BMT. Previous studies have shown reductions in protein C, albumin, factor X and factor VII levels post BMT. Falling protein C levels have been shown to be predictive of severe VOD. These data suggest a role for glutamine in the protection of hepatic function following BMT.

Flow cytometric apoptosis assays indicate different types of endonuclease activity in haematopoietic cells and suggest a cautionary approach to their quantitative use.

Two flow cytometric apoptosis assays, the terminal deoxynucleotidyl transferase (TdT) assay and in situ nick translation (ISNT) assay, were assessed for their ability to quantitate drug-induced apoptosis in CLL lymphocytes. In contrast to HL60 cells, biotinylated dUTP could not be effectively incorporated into apoptotic CLL lymphocytes using exogenous TdT. This suggested that CLL lymphocytes possess a different type of endonuclease that cleaves DNA, leaving blunt or 3' recessed DNA breaks, which are poor substrates for TdT. This possibility was tested using lambda exonuclease, which can convert a blunt or 3' recessed DNA break into a 3' overhang. Apoptotic CLL lymphocytes pre-treated with lambda exonuclease demonstrated increased nucleotide incorporation with TdT. Single-strand DNA breaks are also present in apoptotic CLL lymphocytes, as labelled nucleotides could be incorporated using the in situ nick translation assay. This study suggests that the efficiency of tailing reactions may be limited by the nature of the endonuclease activity in certain cell types and that validation with other parameters is an essential prerequisite to their quantitative use.

Deletions at 11q identify a subset of patients with typical CLL who show consistent disease progression and reduced survival.

Eighty-four patients with typical chronic lymphocytic leukemia (CLL) (by morphological and immunophenotypic criteria) on whom karyotypes were available were studied. Binet stage at diagnosis and follow-up were defined. Survival was calculated from diagnosis. Fifty-one percent of patients had a karyotypic abnormality, the commonest being abnormalities at 13q14 (16%); these patients did not have significantly different survival from patients with normal karyotype. The second commonest abnormality was del(11q) (13%); these patients had significantly worse survival when compared both with patients with normal karyotype (P < 0.0001) and with other patients with karyotypic abnormality (P = 0.0012). All patients with del(11q) had progressed to stage C at follow-up while only 20% of the other patients had shown any disease progression (P < 0.0001). Del(11q) may identify a subset of patients with typical CLL who have worse survival and consistent disease progression and in future may help define a group of patients with CLL who could benefit from earlier or more intensive therapy.

Serum cortisol binding capacity measured with Concanavalin A-Sepharose in patients with a recent inflammatory response.

It has been suggested that elastase released from activated neutrophils degrades cortisol binding globulin. A novel assay for serum cortisol binding capacity was therefore devised and applied to assess whether such degradation was evident in patients showing a recent inflammatory response as indicated by a raised serum C-reactive protein. In 49 patients with evidence (C-reactive protein > 50 mg/l) of a recent inflammatory response, mean serum cortisol binding capacity (288 nmol/l, S.D. = 82.9) was significantly lower (P < 0.05, t test) than in 48 patients (320 +/- 75.8 nmol/l) whose response was quiescent (C-reactive protein < 6 mg/l) or in 49 healthy controls (335 +/- 72.4 nmol/l). Four patients with septic shock had markedly reduced values (167 +/- 49.9 nmol/l) but low values were not restricted to this condition. It is concluded that a population experiencing a recent inflammatory response exhibits reduced serum cortisol binding capacity but a role for elastase in this process remains to be defined.

Psychosocial morbidity and survival in adult bone marrow transplant recipients--a follow-up study.

Previous reports have suggested that the presence of depression and other psychosocial variables may be associated with a poorer prognosis post bone marrow transplantation. Fifty-six patients had both unstructured clinical interviews and interviews using a structured diagnostic instrument (the Composite International Diagnostic Interview) and were followed up a mean of 82.1 months post-BMT. Of these, 42 patients were also interviewed using the Mental Attitude to Cancer Scale. Survival analysis revealed that factors such as depression or the presence of fighting spirit as the predominant coping style did not correlate with length of survival. Our results imply that survival may be more closely related to physical rather than psychosocial factors.