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Risk stratification is critical in the care of patients with myelodysplastic syndromes (MDS). Approximately 10% have a complex karyotype (CK), defined as more than two cytogenetic abnormalities, which is a highly adverse prognostic marker. However, CK-MDS can carry a wide range of chromosomal abnormalities and somatic mutations. To refine risk stratification of CK-MDS patients, we examined data from 359 CK-MDS patients shared by the International Working Group for MDS. Mutations were underrepresented with the exception of TP53 mutations, identified in 55% of patients. TP53 mutated patients had even fewer co-mutated genes but were enriched for the del(5q) chromosomal abnormality (p < 0.005), monosomal karyotype (p < 0.001), and high complexity, defined as more than 4 cytogenetic abnormalities (p < 0.001). Monosomal karyotype, high complexity, and TP53 mutation were individually associated with shorter overall survival, but monosomal status was not significant in a multivariable model. Multivariable survival modeling identified severe anemia (hemoglobin < 8.0 g/dL), NRAS mutation, SF3B1 mutation, TP53 mutation, elevated blast percentage (>10%), abnormal 3q, abnormal 9, and monosomy 7 as having the greatest survival risk. The poor risk associated with CK-MDS is driven by its association with prognostically adverse TP53 mutations and can be refined by considering clinical and karyotype features.

Genetic and epigenetic alterations contribute to the biological and clinical characteristics of myelodysplastic syndromes (MDS), but a role for socioeconomic environment remains unclear. Here, socioeconomic status (SES) for 283 MDS patients was estimated using the Scottish Index of Multiple Deprivation tool. Indices were assigned to quintile categorical indicators ranked from SES1 (lowest) to SES5 (highest). Clinicopathological features and outcomes between SES quintiles containing 15%, 20%, 19%, 30% and 16% of patients were compared. Prognostic scores identified lower-risk MDS in 82% of patients, with higher-risk disease in 18%. SES quintiles did not associate with age, gender, cytogenetics, International Prognostic scores or, in sub-analysis (n = 95), driver mutations. The odds ratio of a diagnosis of refractory anaemia was greater than other MDS sub-types in SES5 (OR 1·9, P = 0·024). Most patients (91%) exclusively received supportive care. SES did not associate with leukaemic transformation or cause of death. Cox regression models confirmed male gender (P < 0·05), disease-risk (P < 0·0001) and age (P < 0·01) as independent predictors of leukaemia-free survival, with leukaemic transformation an additional determinant of overall survival (P = 0·07). Thus, if access to healthcare is equitable, SES does not determine disease biology or survival in MDS patients receiving supportive treatment; additional studies are required to determine whether outcomes following disease-modifying therapies are influenced by SES., (© 2016 John Wiley & Sons Ltd.)

Genital herpes is a common sexually transmitted disease, affecting more than 400 million persons worldwide. It is caused by herpes simplex virus (HSV) and characterized by lifelong infection and periodic reactivation. A visible outbreak consists of single or clustered vesicles on the genitalia, perineum, buttocks, upper thighs, or perianal areas that ulcerate before resolving. Symptoms of primary infection may include malaise, fever, or localized adenopathy. Subsequent outbreaks, caused by reactivation of latent virus, are usually milder. Asymptomatic shedding of transmissible virus is common. Although HSV-1 and HSV-2 are indistinguishable visually, they exhibit differences in behavior that may affect management. Patients with HSV-2 have a higher risk of acquiring human immunodeficiency virus (HIV) infection. Polymerase chain reaction assay is the preferred method of confirming HSV infection in patients with active lesions. Treatment of primary and subsequent outbreaks with nucleoside analogues is well tolerated and reduces duration, severity, and frequency of recurrences. In patients with HSV who are HIV-negative, treatment reduces transmission of HSV to uninfected partners. During pregnancy, antiviral prophylaxis with acyclovir is recommended from 36 weeks of gestation until delivery in women with a history of genital herpes. Elective cesarean delivery should be performed in laboring patients with active lesions to reduce the risk of neonatal herpes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic hematological malignancies characterized by dysplasia, ineffective hematopoiesis and a variable risk of progression to acute myeloid leukemia. Sequencing of MDS genomes has identified mutations in genes implicated in RNA splicing, DNA modification, chromatin regulation, and cell signaling. We sequenced 111 genes across 738 patients with MDS or closely related neoplasms (including chronic myelomonocytic leukemia and MDS-myeloproliferative neoplasms) to explore the role of acquired mutations in MDS biology and clinical phenotype. Seventy-eight percent of patients had 1 or more oncogenic mutations. We identify complex patterns of pairwise association between genes, indicative of epistatic interactions involving components of the spliceosome machinery and epigenetic modifiers. Coupled with inferences on subclonal mutations, these data suggest a hypothesis of genetic "predestination," in which early driver mutations, typically affecting genes involved in RNA splicing, dictate future trajectories of disease evolution with distinct clinical phenotypes. Driver mutations had equivalent prognostic significance, whether clonal or subclonal, and leukemia-free survival deteriorated steadily as numbers of driver mutations increased. Thus, analysis of oncogenic mutations in large, well-characterized cohorts of patients illustrates the interconnections between the cancer genome and disease biology, with considerable potential for clinical application.

Background: Activation of wild-type p53 with the small molecule sirtuin inhibitor Tenovin-6 (Tnv-6) induces p53-dependent apoptosis in many malignant cells. In contrast, Tnv-6 reduces chronic lymphocytic leukaemia (CLL) cell viability with dysregulation of autophagy, without increasing p53-pathway activity., Methods: Here, we have investigated whether a quiescent phenotype (unique to CLL) determines the Tnv-6 response, by comparing the effects of Tnv-6 on activated and proliferating CLL. We further studied if these responses are p53-dependent., Results: Unlike quiescent cells, cell death in activated cultures treated with Tnv-6 was consistently associated with p53 upregulation. However, p53 acetylation remained unchanged, without caspase-3 cleavage or apoptosis on electron microscopy. Instead, cellular ultrastructure and protein profiles indicated autophagy inhibition, with reduced ubiquitin-proteasome activity. In specimens with mutant TP53 cultured with Tnv-6, changes in the autophagy-associated protein LC3 occurred independently of p53. Cells treated with Tnv-6 analogues lacking sirtuin inhibitory activity had attenuated LC3 lipidation compared with Tnv-6 (P0.01), suggesting that autophagy dysregulation occurs predominantly through an effect on sirtuins., Conclusion: These cell cycle and p53-independent anti-leukaemic mechanisms potentially offer novel therapeutic approaches to target leukaemia-sustaining cells in CLL, including in disease with p53-pathway dysfunction. Whether targets in addition to sirtuins contribute to autophagy dysregulation by Tnv-6, requires further investigation.

Tenovin-6 (Tnv-6) is a bioactive small molecule with anti-neoplastic activity. Inhibition of the Sirtuin class of protein deacetylases with activation of p53 function is associated with the pro-apoptotic effects of Tnv-6 in many tumors. Here, we demonstrate that in chronic lymphocytic leukemia (CLL) cells, Tnv-6 causes non-genotoxic cytotoxicity, without adversely affecting human clonogenic hematopoietic progenitors in vitro, or murine hematopoiesis. Mechanistically, exposure of CLL cells to Tnv-6 did not induce cellular apoptosis or p53-pathway activity. Transcriptomic profiling identified a gene program influenced by Tnv-6 that included autophagy-lysosomal pathway genes. The dysregulation of autophagy was confirmed by changes in cellular ultrastructure and increases in the autophagy-regulatory proteins LC3 (LC3-II) and p62/Sequestosome. Adding bafilomycin-A1, an autophagy inhibitor to Tnv-6 containing cultures did not cause synergistic accumulation of LC3-II, suggesting inhibition of late-stage autophagy by Tnv-6. Thus, in CLL, the cytotoxic effects of Tnv-6 result from dysregulation of protective autophagy pathways.

The presence of p53-pathway dysfunction in chronic lymphocytic leukemia (CLL) can be used to identify patients with chemotherapy-refractory disease. Therapeutic responses are known to vary between patients with chemosensitive CLL and may relate to differences in p53-pathway activity. We hypothesized that the magnitude or type of p53-pathway protein expression is heterogeneous in patients with chemosensitive disease and could associate with white cell responses. In this pilot study, changes in p53 and its transcriptional targets, p21/waf1 and MDM2 were analyzed by immunoblotting and densitometry in CLL cells from 10 patients immediately prior to the start of chemotherapy, and after culture for 24 hours (h) with fludarabine (n=7) or chlorambucil (n=3). The in vitro response was also compared to that in vivo in circulating cells pre-treatment, and at 24h and 96h of chemotherapy. Disease responses were evident in all patients after the first treatment-cycle. Significant p53 induction was observed in CLL cells treated in vitro and in vivo. Greater heterogeneity in the expression-intensity was observed in vivo (σ2=45.15) than in vitro (σ2=1.33) and the results failed to correlate (r(2)=0.18, p=0.22). p21/waf1 and MDM2 expression-profiles were also dissimilar in vitro and in vivo. Higher in vivo (but not in vitro) responses associated with changes in white cell count (p=0.026). Thus, heterogeneity of p53-pathway activity exists in chemosensitive CLL; in unselected patients, in vivo changes do not correlate with those in vitro, but may associate with post-treatment white cell responses.

In a previous study, we identified somatic mutations of SF3B1, a gene encoding a core component of RNA splicing machinery, in patients with myelodysplastic syndrome (MDS). Here, we define the clinical significance of these mutations in MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). The coding exons of SF3B1 were screened using massively parallel pyrosequencing in patients with MDS, MDS/MPN, or acute myeloid leukemia (AML) evolving from MDS. Somatic mutations of SF3B1 were found in 150 of 533 (28.1%) patients with MDS, 16 of 83 (19.3%) with MDS/MPN, and 2 of 38 (5.3%) with AML. There was a significant association of SF3B1 mutations with the presence of ring sideroblasts (P < .001) and of mutant allele burden with their proportion (P = .002). The mutant gene had a positive predictive value for ring sideroblasts of 97.7% (95% confidence interval, 93.5%-99.5%). In multivariate analysis including established risk factors, SF3B1 mutations were found to be independently associated with better overall survival (hazard ratio = 0.15, P = .025) and lower risk of evolution into AML (hazard ratio = 0.33, P = .049). The close association between SF3B1 mutations and disease phenotype with ring sideroblasts across MDS and MDS/MPN is consistent with a causal relationship. Furthermore, SF3B1 mutations are independent predictors of favorable clinical outcome, and their incorporation into stratification systems might improve risk assessment in MDS.

Quantitative mass spectrometry-based proteomics is a vital tool in modern life science research. In contrast to the popularity of approaches for relative protein quantitation, the widespread use of absolute quantitation has been hampered by inefficient and expensive production of labeled protein standards. To optimize production of isotopically labeled standards, we genetically modified a commonly employed protein expression Escherichia coli strain, BL21 (DE3), to construct an auxotroph for arginine and lysine. This bacterial strain allows low-cost, high-level expression of fully labeled proteins with no conversion of labeled arginine to proline. In combination with a fluorescence-based quantitation of standards and nontargeted LC-MS/MS analysis of unfractionated total cell lysates, this strain was used to determine the copy number of a post-translational modifier, small ubiquitin-like modifier (SUMO-2), in HeLa, human sperm, and chronic lymphocytic leukemia cells. By streamlining and improving the generation of labeled standards, this production system increases the breadth of absolute quantitation by mass spectrometry and will facilitate a far wider uptake of this important technique than previously possible.

Consideration of iron-chelation (IC) in transfusion-dependent patients is recommended in most clinical-practice guidelines on myelodysplastic syndromes (MDS). The financial impact of IC on health-care systems is predicted through economic modeling, but an analysis based on actual prevalence is lacking. Here, we have investigated the potential drug-costs and need for IC in a cohort of 189 United Kingdom-based MDS patients diagnosed from 2000 to 2010. Patients with low or intermediate-1 IPSS scores were identified as eligible for IC if ≥24 red cell units (RCU) had been transfused over 12 consecutive months or the transfusion-intensity averaged ≥2 RCU per month. Drug-costs were calculated from the time patients qualified for IC until death or last follow-up. In 159 patients with low/intermediate-1 MDS, survival was superior with a low IPSS score (P = 0.014), age <70 years (P = 0.043), transfusion-independence at diagnosis (P = 0.0056) and transfusion-intensity of <2 RCU per month (P = 0.009). Reflecting the time elapsed since diagnosis, longer survival was observed with a cumulative red cell load of ≥75 U (P = 0.046). By logistic-regression analysis, transfusion-intensity independently predicted survival (P = 0.0035) in low and intermediate-1 risk MDS patients. Forty-one patients fulfilled criteria for consideration of IC. Of these, 6 patients died within 1 month; 35 patients survived for a median of 16 months (range 1-61). Had patients commenced IC, the anticipated drug-costs alone would have been ~$526,880-$2,064,800 over 10 years. The lack of association between cumulative transfusion-load and survival calls for a prospective evaluation of the cost-utility of IC in patients surviving long-term, to enable evidence-based recommendations in MDS management., (Copyright © 2011 Wiley-Liss, Inc.)

Following sciatic nerve transection in adult rats, a proportion of injured dorsal root ganglion (DRG) neurons die, through apoptosis, over the following 6 months. Previous studies showed that axotomy and neurotrophin-3 administration may have effects on expression of neurotrophins and their receptors in DRG. In the current study, the fourth and fifth lumbar DRGs of rats were examined 2 weeks after right sciatic nerve transection and ligation. The effects of axotomy and systemic NT-3 treatment on neuronal genes were investigated by microarray. The results demonstrated that bone morphogenetic protein (BMP) and Janus protein tyrosine kinase signaling pathways are induced in axotomized DRG, and PI-3 kinase and BMP pathways and genes controlling various cellular functions were induced after axotomy and NT-3 administration.

The detection of Philadelphia-negative (Ph(neg)) cells with non-random karyotypic abnormalities after tyrosine kinase inhibitor (TKI) therapy of chronic myeloid leukaemia (CML) can be associated with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). To our knowledge, however, there have been no studies on variables influencing the risk of MDS/AML in patients with specific Ph(neg) karyotypes. We systematically examined studies reporting -7 or del(7q) within Ph(neg) cells in TKI-treated CML patients, and abstracted clinical and cytogenetic data from individual reports into a standardized format for further analysis. Of 53 patients, 43 had Ph(neg) -7 clones [as the sole abnormality (-7(sole)) in 29, or with other clones (-7(dual)) in 14], and del(7q) was present in 10. A total of 16/51 evaluable patients, all with -7, transformed to MDS/AML. Transformation was more frequent (15/16 patients) within 6 months of Ph(neg) -7 detection rather than subsequently (P < 0.0001). At first detection after TKI therapy, Ph(neg) abnormal clones comprised ≥50% of Ph(neg) cells in a greater proportion of patients with -7 than del(7q) (P = 0.035). Upon comparing -7(sole) and -7(dual), the latter was likely to be transient (P = 0.004), and AML was frequently observed with persistent -7 clones (P = 0.03). By logistic regression analysis (n = 36), clone size (P = 0.017), time-to-detection longer than 15 months (P = 0.02), and CML response (P = 0.085) were associated with MDS/AML. Validation of these novel associations in registry-based studies will help develop predictive criteria that define the MDS/AML risk in individual patients., (Copyright © 2011 Elsevier Inc. All rights reserved.)

p53 gene expresses several protein isoforms modulating p53-mediated responses through regulation of gene expression. Here, we identify a novel p53 isoform, Δ160p53, lacking the first 159 residues. By knockdown experiments and site-directed mutagenesis, we show that Δ160p53 is encoded by Δ133p53 transcript using ATG160 as translational initiation site. This hypothesis is supported by endogenous expression of Δ160p53 in U2OS, T47D and K562 cells, the latter ones carrying a premature stop codon that impairs p53 and Δ133p53 protein expression but not the one of Δ160p53. Overall, these results show that the Δ133p53 transcript generates two different p53 isoforms, Δ133p53 and Δ160p53., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In the months following transection of adult rat peripheral nerve some sensory neurons undergo apoptosis. Two weeks after sciatic nerve transection some neurons in the L4 and L5 dorsal root ganglia begin to show immunoreactivity for nestin, a filament protein expressed by neuronal precursors and immature neurons, which is stimulated by neurotrophin-3 (NT-3) administration. The aim of this study was to examine whether NT-3 administration could be compensating for decreased production of neurotrophins or their receptors after axotomy, and to determine the effect on nestin synthesis. The levels of mRNA in the ipsilateral and contralateral L4 and L5 dorsal root ganglia were analyzed using real-time polymerase chain reaction, 1 day, 1, 2 and 4 weeks after unilateral sciatic nerve transection and NT-3 or vehicle administration via s.c. micro-osmotic pumps. In situ hybridization was used to identify which cells and neurons expressed mRNAs of interest, and the expression of full-length trkC and p75NTR protein was investigated using immunohistochemistry. Systemic NT-3 treatment increased the expression of brain-derived neurotrophic factor, nestin, trkA, trkB and trkC mRNA in ipsilateral ganglia compared with vehicle-treated animals. Some satellite cells surrounding neurons expressed trkA and trkC mRNA and trkC immunoreactivity. NT-3 administration did not affect neurotrophin mRNA levels in the contralateral ganglia, but decreased the expression of trkA mRNA and increased the expression of trkB mRNA and p75NTR mRNA and protein. These data suggest that systemically administered NT-3 may counteract the decrease, or even increase, neurotrophin responsiveness in both ipsi- and contralateral ganglia after nerve injury.

The pathogenesis of acute myeloid leukemia (AML) involves the cooperation of mutations promoting proliferation/survival and those impairing differentiation. The RAS pathway has been implicated as a key component of the proliferative drive in AML. We have screened AML patients, predominantly younger than 60 years and treated within 2 clinical trials, for NRAS (n = 1106), KRAS (n = 739), and HRAS (n = 200) hot-spot mutations using denaturing high-performance liquid chromatography or restriction fragment length polymorphism (RFLP) analysis. NRAS mutations were confirmed in 11% of patients (126/1106) and KRAS mutations in 5% (39/739). No HRAS mutations were detected in 200 randomly selected samples. Codons most frequently mutated were N12 (43%), N13 (21%), and K12 (21%). KRAS mutations were relatively overrepresented in French-American-British (FAB) type M4 (P < .001). NRAS mutation was over-represented in the t(3;5)(q21 approximately 25;q31 approximately q35) subgroup (P < .001) and underrepresented in t(15;17)(q22;q21) (P < .001). KRAS mutation was overrepresented in inv(16)(p13q22) (P = .004). Twenty-three percent of KRAS mutations were within the inv(16) subgroup. RAS mutation and FLT3 ITD were rarely coexistent (14/768; P < .001). Median percentage of RAS mutant allele assayed by quantitative RFLP analysis was 28% (N12), 19% (N13), 25% (N61), and 21% (K12). RAS mutation did not influence clinical outcome (overall/disease-free survival, complete remission, relapse rate) either for the entire cohort or within cytogenetic risk groups.

Somatropin, human growth hormone (hGH), is an unstable protein, posing challenging problems for its formulation and long-term stability. Since hGH formed insoluble adducts with heparin our aim was to evaluate heparin as a stabilizing agent for the drug. These adducts were characterized by particle diameter, tertiary structure variations and release studies. Studies were also carried out to determine the stability of hGH in the presence and absence of heparin by an interfacial denaturation method and real-time stability studies by measuring hGH activity and particle diameter. Moreover, biological activity of hGH and hGH/UH (unfractionated heparin) adducts was identified by daily subcutaneous injections to hypophysectomized rats. There was a decrease in mean hydrodynamic particle diameter of hGH/UH adducts with increased pH (54.4 to 12.2 nm from pH 3 to pH 7) indicating that the adducts were either dissociating or dissolving at high pH. Furthermore, second-derivative spectroscopy indicated that complexation of hGH with heparin did not cause a major disruption in the tertiary structure of hGH but decreased the hydrophilic environment around the tyrosine residues. Release of hGH from hGH/UH adducts was pH and ionic strength dependent with the highest release at pH 8 (93%) and lowest release at pH 3 (0%) over the first hour. Interfacial denaturation methods indicated that vortex agitation over 120 s resulted in no change in the optical density of hGH/UH adducts compared with a substantial increase for hGH alone at pH 6.8. Real-time stability studies over 93 days demonstrated that hGH/UH adducts at both pH 3 and 7 with an excess of heparin produced the highest percent of active hGH remaining in the solution at 4 degrees C and 37 degrees C. The higher stability of hGH/UH adducts with excess heparin compared with the stoichiometric ratio was also confirmed by particle size measurements during storage. The biological activity of these adducts was comparable with hGH alone by weight-gain studies in hypophysectomized rats. The findings suggest the value of using hGH/heparin adducts to stabilize the protein.

Previous work has shown that administration of the neurotrophin NT-3 intrathecally or to the proximal stump can prevent axotomy-induced sensory neuron loss and that NT-3 can stimulate sensory neuron differentiation in vitro. We have examined the effect of axotomy and systemic NT-3 administration on neuronal loss, apoptosis (defined by morphology and activated caspase-3 immunoreactivity), and nestin expression (a protein expressed by neuronal precursor cells) in dorsal root ganglia (DRG) following axotomy of the adult rat sciatic nerve. Systemic administration of 1.25 or 5 mg of NT-3 over 1 month had no effect on the incidence of apoptotic neurons but prevented the overall loss of neurons seen at 4 weeks in vehicle-treated animals. Nestin-immunoreactive neurons began to appear 2 weeks after sciatic transection in untreated animals and steadily increased in incidence over the next 6 weeks. NT-3 administration increased the number of nestin-immunoreactive neurons at 1 month by two- to threefold. Nestin-IR neurons had a mean diameter of 20.78 +/- 2.5 microm and expressed the neuronal markers neurofilament 200, betaIII-tubulin, protein gene product 9.5, growth associated protein 43, trkA, and calcitonin gene-related peptide. Our results suggest that the presence of nestin in DRG neurons after nerve injury is due to recent differentiation and that exogenous NT-3 may prevent neuron loss by stimulating this process, rather than preventing neuron death., (Copyright 2005 Wiley-Liss, Inc.)

Extracts obtained from three Nigerian Sterculiaceae plants: Cola accuminata, C. nitida and C. milleni were screened for anti-mycobacterium properties using a slow growing Mycobacterium bovis ATCC 35738 (designated BCG Mexican and known to have some virulence in mouse and guinea pig) at 1000 microg/ml using the radiometric (BACTEC) method. The extracts were also tested against six fast growing ATCC strains of M. vaccae using the broth microdilution method. The methanol extracts from both leaves, stem bark and root bark of Cola accuminata and from the leaves and stem bark of C. nitida and C. milleni were not active at the highest concentration of 1000 microg/ml. Only the methanol extract of root bark for both C. nitida and C. milleni were found to be potent against both M. bovis and strains of M. vaccae. The minimum inhibitory concentration (MIC) of C. nitida against M. bovis is 125 microg/ml while the MIC of C. milleni against M. bovis is 62.5 microg/ml after at least 6 days of inhibition with growth index (GI) units lesser than or equal to the change in GI units inoculated with a 1/100 of the BACTEC inoculum for a control vial. The minimum inhibitory concentration of C. milleni against the six ATCC strain of M. vaccae ranged from 62.5 microg/ml to 250 microg/ml while for C. nitida ranged from 500 microg/ml to above 1000microg/ml. Evidently, C. milleni has the highest inhibitory activity against both M. bovis and strains of M. vaccae used. Rifampicin, the positive control used has strong activity against M. bovis at the tested concentration of 5 microg and 10 microg/ml and 4 to 8 microg/ml against the six strains of M. vaccae., (Copyright 2004 John Wiley & Sons, Ltd.)

A spontaneous autosomal recessive mutation was identified in the Sprague-Dawley rat strain with an early onset sensory neuropathy. The main clinical features of the mutation (mutilated foot, mf ), detectable shortly after birth, include ataxia, insensitivity to pain and foot ulceration. The pathological features include a severe reduction in the number of sensory ganglia and fibres. This mutant is therefore an excellent model for human hereditary sensory neuropathies. Here, we demonstrate that the mf locus maps to the distal end of rat chromosome 14, a region syntenic to human 2p13-p16 and proximal mouse 11. Sequence analysis of four candidate genes in this interval revealed a 1349G>A mutation in the chaperonin (delta) subunit 4 (Cct4) gene associated with the mf mutant. This change resulted in the substitution of a highly conserved cysteine for tyrosine at amino acid 450. Although we did not identify a mutation in the human CCT4 gene in a set of HSN patients, this result clearly demonstrates the pathological consequences of a defect in Cct4, a subunit of CCT (cytosolic chaperonin-containing t-complex peptide-1), involved in folding tubulin, actin and other cytosolic proteins. This is the first report of a mutation in a molecular chaperonin causing a hereditary neuropathy and raises the possibility that mis-folding proteins may be a cause of this group of neuropathies.

Microvasculitis may play a greater part in the pathogenesis of paraproteinaemic neuropathies than is generally recognised, producing tissue destruction by convergent immune and physical mechanisms. We present a patient with a clinical syndrome of mononeuritis multiplex and a circulating IgM lambda paraprotein, in whom bone marrow aspiration revealed a lymphoplasmacytoid lymphoma. Microvasculitic changes were present in the first nerve biopsy, and the second showed extensive destruction of neural architecture and deposition of IgM-related material. A 2-stage pathogenic cascade is postulated and explored with a review of the relevant literature.

Following permanent transection of the adult rat sciatic nerve, sensory neuron apoptosis in the contributing L4 and L5 dorsal root ganglia can be observed for at least 6 months afterwards. To establish the profile of any sensory neuron apoptosis and loss over time when axonal regeneration is allowed, serial sections of L4 and L5 ganglia were examined and the neurons counted using a stereological technique 1, 2 and 3 months after crushing the right sciatic nerve at mid-thigh level. Our results show that an identical degree of sensory neuron loss and apoptosis occurs 1 month after crush as at 1 month after permanent transection. However, at 3 months no neurons undergoing apoptosis could be observed and no significant loss could be detected in the ipsilateral ganglia when compared to unoperated controls. One explanation was a neuronal replacement mechanism, which was investigated by administering bromodeoxyuridine to rats for 1 month after sciatic nerve transection or crush, prior to detection using immunohistochemistry on sections of their ganglia after 2 months. The presence of bromodeoxyuridine in the nuclei of occasional cells that would be counted as neurons on the basis of size and morphology indicates that a process of apparent neurogenesis may underlie the profile of sensory neuron loss after axotomy.

PS4alpha is a high molecular weight peptidoglycan extracted from Mycobacterium vaccae, which has demonstrated considerable antineoplastic activity in-vivo without apparent toxicity. Available fortesting in only small quantities, a sensitive in-vivo method for measuring pulse and breathing rates in cannulated rats was applied to this compound at doses of 5, 50 and 500 microg kg(-1). Various parameters (mean arterial pressure, maximum transpulmonary pressure, compliance, heart rate, minute volume, respiratory rate and tidal volume) were followed for up to 1 h and demonstrated no significant deviation in the baseline values obtained before injection. This compound at doses up to 500 microg kg(-1) had no apparent acute toxicity in rats, but chronic effects at this and higher doses have to be determined by more conventional toxicological methods before proceeding to evaluate PS4alpha as an antineoplastic agent.

The release mechanism of high molecular weight fluorescein isothiocyanate dextrans (FITC-dextrans) from HPMC hydrogel matrices was studied. An anomaly was noted in the release behaviour of a series of high molecular weight FITC-dextrans from a tablet formulation designed to float in stomach contents. The tablets contained sodium bicarbonate and hydroxypropylmethyl cellulose (HPMC) in a cetyl alcohol matrix. When hydrated in an acid medium, this tablet consisted of a mixed solid with a viscous surface layer containing carbon dioxide bubbles through which the active ingredient (FITC-dextran) was released into the aqueous environment. However, it was observed that, above a critical molecular weight (approx. 65 kDa), the FITC-dextran was only released into the medium by an erosion-type mechanism, whereas, below this value, both diffusion and erosion processes took place. The key constraint appeared to be the apparent gel pore-size of the hydrated HPMC that was approximately 12 nm in diameter, irrespective of the molecular weight of the HPMC samples evaluated. It was concluded that FITC-dextran release was controlled by both FITC-dextran molecular weight and the HPMC hydrogel structure.

Background and Objectives: Glutathione S-transferases (GSTs) are phase II metabolizing enzymes which catalyze the conjugation of glutathione (GSH) to electrophilic substrates and possess selenium-independent glutathione peroxidase activity. The GST enzyme family includes the cytosolic isoforms GST-alpha, mu (GSTM), pi (GSTP), theta (GSTT) and sigma (GSTS). GSTT1, P1 and M1 are polymorphic and altered polymorphic frequency of genes encoding these proteins has been suggested as a potential risk factor for the development of hematopoietic malignancies. Overexpression of GSTs has also been implicated in chemotherapeutic drug resistance. This study was undertaken to elucidate the potential functional relevance of these genetic polymorphisms in hematopoiesis., Design and Methods: GST genotype of 14 hematopoietic cell lines was determined by polymerase- chain-reaction (PCR). Gene expression of GSTs in a cell line was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on TaqMan 7700 and by semi-quantitative RT-PCR. Cytosolic GST protein expression was detected by Western blot. GST conjugation activity was assayed using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate., Results: GSTP1 expression was higher than other GSTs in 13/14 cell lines and paralleled CDNB conjugation activity. GSTP1 and GSTM1 predominated in lymphoid lines whilst T1 expression was relatively greatest in erythroid lines but was absent in 7/12 non-null lines. GSTT2 was expressed in only 3/4 lines. The 3 cell lines which expressed GSTA1 were all erythroid., Interpretation and Conclusions: Glutathione S-transerases showed differential lineage expression in hematopoietic cell lines. This implies a greater cytoprotective role for GSTT1 and GSTA1 in erythroid cells and GSTM1 in lymphoid cells. We postulate that inherited gene deletion of GSTT1 and M1 may produce increased genotoxic susceptibility for erythroid and lymphoid cell respectively, following exposure to xenobiotics that are substrates for these enzymes.

A polysaccharide around 3.6 kDa has been identified as the major carbohydrate moiety of a antineoplastic protein-polysaccharide complex (PS4A) obtained by boiling intact cells of Mycobacterium vaccae in water. 1H and 13C NMR spectra of this polysaccharide suggested it was a highly homogeneous polymer composed substantially of one monomer, probably an alpha-linked O-methylated mannose. Comparison of the COSY spectra of the original and acetylated polymer indicated that the glycosidic linkage and the methyl ether were interchangeable, at O-3 and O-4. Further study demonstrated that the benzyolated hydrolysate of the polymer was 1,2,4,6-tetra-O-benzoyl-3-O-methyl-beta-mannopyranose. The hydrolysate was 3-O-methyl-alpha, beta-mannopyranose and the polymer was therefore poly-alpha-(1-->4)-linked 3-O-methyl-D-mannopyranose. This conclusion was further confirmed with an authentic sample of the monomer, which had spectral data identical to those of the hydrolyzate and co-eluted from an ion-exchange HPLC with the major sugar in the hydrolysate.

Adult dorsal root ganglion neurons express oligosaccharides conjugated to lipids that may be involved in cell-cell recognition, and consequently in the laminar organisation of their central terminations. This paper describes an immunohistochemical study of the developmental expression of 2 lactoseries (LA4 and LD2) and 1 globoseries (SSEA4) oligosaccharide conjugates in rats from embryonic d 19 to postnatal d 60. The expression of calcitonin gene related peptide and the growth associated protein GAP43 was also examined for comparative purposes. We found that these oligosaccharide conjugates begin to be expressed after birth, suggesting that they may be involved in maturation of the central or peripheral terminations, rather than axonal guidance.

Although it has been claimed that nanoparticles can be produced from gelatin, a naturally occurring polypeptide, the commercial conversion of animal collagen to gelatin results in a heterogeneous product with a wide molecular-weight range. This is probably responsible for the widely observed variation in the experimental conditions required for nanoparticle formation. In this study, 0.2% w/v aqueous B225 gelatin solutions were incubated under various conditions of time, temperature, pH and ethanol concentration and characterized by both size-exclusion high-performance liquid chromatography (HPLC) and dynamic light scattering. Gelatin was shown to be denatured when the temperature was increased to 37 degrees C (approx.) and the rate of renaturation was optimized over the temperature range 7-20 degrees C at pH 5.0, equivalent to the isoelectric point (IEP). The molecular-weight profile remained unchanged at 37 degrees C (approx.) in the pH range 5-7. When the gelatin solutions were mixed with ethanol, higher-molecular-weight fractions (microgel, delta and zeta fractions, all with molecular weights > 700 kDa) precipitated at ethanol concentrations lower than those required to precipitate the lower molecular weight material ( < 700 kDa), with maximum precipitation occurring close to the isoelectric point (pH 5.0). The molecular weight profile of gelatin in solution is evidently critically affected in a time-dependent manner by both pH and temperature. These two factors influence the noncovalent interactions responsible for the molecular structure of gelatin. The molecular weight profiles, in turn, affect the phase behaviour of gelatin in hydroalcoholic solutions. Systematically investigating the effect of time, temperature, pH and ethanol concentration on the molecular-weight-distribution profile of a gelatin solution enabled a robust method to be developed for the preparation of colloidal dispersions of non-aggregated gelatin nanoparticles 220-250 nm in diameter. This contrasts with the multiparticulate aggregates produced by earlier literature methods.

Background: Bacillus Calmette-Guérin (BCG) vaccine is a non-specific immunostimulant which has been used clinically in the treatment of melanoma. In this communication, the antimelanoma activity of BCG was related to its fibronectin-binding properties and mimicked using gelatin nanoparticles., Materials and Methods: The fibronectin-binding properties of aqueous gelatin solutions, gelatin nanoparticles, BCG vaccine, and PS1 (a glucan extracted from Tice BCG) were compared by an enzyme-linked immunosorbent assay and their ability to suppress murine B16-F0 melanoma in vivo investigated., Results: Aqueous gelatin solutions, gelatin nanoparticles and BCG all bound to fibronectin in vitro. The immunostimulant PS1 did not. In vivo, BCG and gelatin nanoparticles suppressed melanoma growth while PS1 and aqueous gelatin solutions had no effect., Conclusions: The antimelanoma activity of BCG is not due to the associated immunostimulatory glucan but can be correlated to its fibronectin-binding properties. Since solutions of gelatin have no effect whereas nanoparticles produce total suppression, this suggests a relationship between the volume of the fibronectin-binding entities and their antitumour activity. Thus, gelatin nanoparticles may represent an attractive alternative to the use of BCG vaccine in melanoma treatment.

Although heat-killed suspensions of Mycobacterium vaccae have been tested clinically against tuberculosis and cancer, from a pharmaceutical perspective it would be advantageous to utilize isolated active components rather than the heat-degraded bacterial materials. In our laboratory we have isolated from M. vaccae a number of high-molecular-weight proteoglycans with considerable immunological and antineoplastic activity. The structure of one of these, PS4A, obtained by extraction with boiling water, seems to consist of a basic unit with a 20-kDa protein core to which are attached glucans and O-methylated 4-kDa polysaccharides. The molecular weight is (approx.) 50 kDa, but because of self-association, that of the recovered high-molecular-weight fraction is greater than 150 kDa. A similar, but even larger, molecule (PS4alpha, MW approximately 20 MDa) is obtained by cold extraction with 8 M urea. Both are active in-vivo against an S-180 murine sarcoma model but have no activity in-vitro, suggesting an antitumour effect involving activated macrophages. For this reason gelatin nanoparticles are unsuitable as a vehicle but chitosan seemed to be a promising alternative. In this report we describe the production of stable 600-700-nm diameter nanoparticles of chitosan without organic solvents. Adsorption and release of bovine serum albumin seemed to be affected by the charge of the two reactants and at high doses not all adsorbate was released. PS4A, because of structural and compositional differences, had to be loaded on to the chitosan by freeze drying a suspension of the nanoparticles in a solution of the drug. After a rapid (burst) release phase, the rate of release into water was steady for the next 4 h, but not all the drug was released. In-vivo it was evident that PS4A and PS4alpha were equally active in solution or when formulated in the chitosan nanoparticles. These results show that chitosan nanoparticles, readily prepared without the use of organic solvents, are a suitable vehicle for the delivery of these immunostimulants from M. vaccae; the formulations might find application as antitumour agents.

Following permanent transection of their peripheral axons, a proportion of adult rat dorsal root ganglion neurons undergo programmed cell death (apoptosis) over a period of months. The underlying causes of this neuron loss are unclear, but may involve the interruption of the supply of target-derived neurotrophic factors, the replacement of which could prevent this loss from occurring. To investigate whether the administration of neurotrophic factors can prevent the dorsal root ganglion neuron death in adults, a 1 mg/ml solution of ciliary neurotrophic factor or of NT-3 was applied via a silicon reservoir to the proximal stump after unilateral sciatic transection at mid-thigh level. The incidence of apoptotic neurons and neuronal loss in the L4 and L5 ganglia ipsilateral to sciatic nerve transection when compared with the contralateral ganglia was then measured 1 month later. This was assessed by examining serial sections of ganglia for neurons undergoing apoptosis and expressing the total counted as a percentage of the total number of neurons estimated using a stereological neuron counting technique. Our results show that NT-3 administration significantly reduced the incidence of apoptotic neurons and prevented neuron loss, while CNTF had no effect on either parameter., (Copyright 1999 Academic Press.)

A mixture of water-soluble protein-polysaccharides (PS4A) was isolated by boiling intact cells of Mycobacterium vaccae, a fast growing mycobacterium. Sephadex G-75 column chromatography of the crude extract separated the biologically active high molecular weight (> 50 kDa) fraction (in the void volume) from the low molecular weight degradation products. Compositional analysis demonstrated that PS4A contained protein and polysaccharide in a ratio of approximately 1.5 to 1, but no lipids were detected. The antineoplastic activity was tested in vivo by a S-180 murine sarcoma model using female CFW mice. The immunostimulating activity was tested in vitro using murine peritoneal macrophages isolated from BALB/C mice. The results demonstrated that PS4A significantly decreased tumor incidence in vivo and produced activation of murine peritoneal macrophages. However, the antineoplastic activity was only attributable to the high molecular weight fraction of the protein-polysaccharide complex. The low molecular weight fraction had no antineoplastic activity in vivo despite stimulation of TNF-alpha production in vitro. In vitro experiments also demonstrated that although all PS4A components significantly increased TNF-alpha production by macrophages, the high molecular weight fraction stimulated more IL-1 production, indicating a better immunostimulating activity.

Bacillus Calmette-Guérin (BCG) vaccine, developed originally for the prophylaxis of tuberculosis, is a potent immunostimulant used to treat superficial bladder carcinoma in man. The aim of this study was to compare the molecular weight and self-association properties of an antineoplastic glucan (PS1A1) extracted from BCG vaccine as determined by different techniques including diffusion, light-scattering and chromatographic methods. In the diffusion experiments, a semi-empirical relationship was derived between the effective diffusion coefficients, Dp, and the weight-average molecular weights, Mw, of several dextrans used as standards, according to the equation Dp = 2.233 x 10(-6) x Mw(-0.66). On the basis of this relationship, the molecular weight of PS1A1 was found to be 57.4 kDa, although, unexpectedly, membrane association was high, most probably because of molecular branching. In the light-scattering experiment it was observed that, unlike dextran, PS1A1 undergoes concentration-dependent multimerization in water. However, the molecular weight of PS1A1 in 0.1 M sodium chloride ranged from 60 to 68 kDa, with a mean of 65 kDa, over the same concentration range. This value was in agreement with the molecular weight determined for PS1A1 by gel-filtration chromatography in previous studies, suggesting that 65 kDa represents the approximate monomeric size of the unassociated molecule. Thus, it was evident that the aggregation was suppressed by electrolyte. Elemental analysis by X-ray fluorescence showed that PS1A1 contained carbon, oxygen, hydrogen and phosphorus, indicating that hitherto unobserved ionized phosphate groups might promote electrostatic interactions.

Gelatin binds to fibronectin with a high affinity although the fibronectin-binding components have not been located. Fibronectin plays an important role in tumor cell metastasis and gelatin may have a profound effect on the metastatic process. In this study, fractionated acid-washed gelatin was cleaved with trypsin and resultant peptides fractionated by fibronectin-Sepharose affinity chromatography. After further purification using size exclusion HPLC and then reverse-phase HPLC, two unique peptides were obtained and sequenced. The binding affinities of these two peptides to fibronectin were evaluated by an ELISA method developed during this study and compared with the gelatin. Both possessed significantly higher binding affinities to fibronectin than gelatin alone.

The polysaccharidic integument surrounding growing cells of attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine, Connaught substrain, can be removed with non-specific proteases. After 5 weeks incubation at 37 degrees C in Middlebrook 7H-9 medium, the collected cells were incubated with pronase and the integument and cells separated by centrifugation in a Ficoll-Histopaque preparation. After washing and drying, the detached integument accounted for 65% w/w of the original dried cell mass. Like the original cellular material, the detached integument manifested antineoplastic activity against a murine sarcoma model in vivo. Solubilization of the otherwise insoluble integument by boiling in water or by digestion in 8 M urea significantly enhanced activity of the integument itself, by 125 and 1,100 times, respectively. Integument extracts were shown to contain mainly glucose, with smaller quantities of other sugars, consistent with the presence in BCG of high molecular weight glycans, as previously reported. It is suggested that most, if not all, of the antineoplastic activity of BCG can be accounted for by the activity associated with the high-molecular weight polysaccharidic glycans which constitute the cellular integument.

Background: Attenuated Mycobacterium bovis, Bacillus Calmette Guerin, BCG vaccine, is a general immune stimulant and is now an approved clinical treatment for superficial bladder cancer. Isolation and characterization of a series of complex polysaccharides (glycans) from BCG and other mycobacteria has shown that these materials are remarkably heat stable and have considerable in vivo activity against a number of animal cancer models. This present communication describes the testing of a glycan, PS1, obtained from the Tice substrain of BCG against the hormonal dependent human breast cancer cell line MCF-7 and the hormonally independent BT-20 line, using 5-fluorouracil (5-FU) as a positive control., Materials and Methods: The PS1 was obtained by methods previously described. Cells were obtained from the American Type Culture Collection (Rockville, MD) and athymic nu/nu mice from Frederick (MD). The cells were implanted into the flanks of 20g female nude mice (n = 10). After two weeks, volumes of phosphate buffered saline (control), 5-FU (positive control) or PS1 solutions were injected and the tumor growth rates followed for up to six weeks., Results: The 5-FU was effective in slowing tumor growth of both tumors. The MCF-7 cell line was markedly affected by the PS1, especially in the presence of estradiol. The BT-20 cell line was only marginally affected by PS1, with or without estradiol., Conclusions: Since PS1 is known to have macrophage stimulating activity and nude mice are deficient in both T-cells and natural killer cells, the mechanism of activity is postulated to involve MHC-1 antigen secretion by the hormonal-dependent tumor cells, enhanced in the presence of hormone. These cells are then actively identified and destroyed by local macrophages.

The mycobacterial cell wall visualized by transmission electron microscopy (TEM) of thin sections of resin-embedded specimens is generally believed to consist of an electron-dense peptidoglycan, an electron-transparent arabinogalactanmycolate layer and an electron-dense outer layer (OL). In addition, a pseudocapsule known as the 'electron-transparent zone' (ETZ) has been observed after phagocytosis of mycobacteria by macrophages. TEM of thin sections of Mycobacterium bovis BCG, Tice substrain, revealed an OL bilayer, each of which measured 2-4 nm in diameter. The intermediate electron-transparent layer varied from 1 to about 250 nm in diameter and appears to be a previously observed oxygen-dependent amorphous integument that consists of hot water-extractable neutral polysaccharides, especially a recently characterized alpha glucan, comprising about 12% of the dry cell weight. This and other recent studies of BCG have revealed cell-surface features that may provide a better understanding of the outer mycobacterial cell envelope.

The purpose of this investigation was to evaluate the application of high molecular weight, insoluble collagen as a carrier material for proteins. Matrices were formulated and their behavior in buffer solution was investigated with focus on swelling and inner structure. Cross-linking with glutaraldehyde was introduced prior to the formation of the devices and its influence characterized. In addition, the enzymatic degradation process was studied and release experiments with systems loaded with fluorescent-labeled bovine serum albumin were carried out. Insoluble collagen matrices were characterized by intensive swelling in buffer resulting in development of a coarse porous character. Cross-linking strongly reduced the water penetration, leading to denser structures of the swollen devices. The continuous enzymatic degradation of the disk-shaped matrices by collagenase followed the kinetics of an heterogeneous enzymatic process with hindrance of proteolysis by the addition of glutaraldehyde. Release studies demonstrated that large amounts of model protein were held in the matrices with increased cross-linking degree. In presence of collagenase a prolonged release of the trapped protein over several days by matrix cleavage could be achieved. Insoluble collagen can be effective as a carrier material for proteins with an in vitro release characteristic by both diffusion-controlled and enzymatic degradation mechanisms. Cross-linking at the stage of preparing the aqueous dispersion offers an alternative to subsequent cross-linking processes.

Using immunocytochemical and morphometric techniques, the localisation of three neuronal oligosaccharide antigens (two lactoseries and one globoseries oligosaccharide) were studied in the spinal cord and dorsal root ganglia of adult rats following unilateral crushing or transection of the sciatic nerve. The expression of CGRP and GAP43 was also studied for comparison. We found that following transection of the nerve the expression of lactoseries oligosaccharides and CGRP was permanently depressed, whilst that of the globoseries antigen (SSEA4) was unaffected. However following crush trauma and subsequent regeneration after 2 months, only the expression of one lactoseries antigen, LA4 remained significantly depressed. Our results suggest that different subsets of sensory neurons vary in the rate of reaction to injury and that one subset of neurons expressing a lactoseries oligosaccharide antigen is particularly susceptible to axotomy-induced changes. Furthermore neurons expressing the globoseries oligosaccharide antigen SSEA4 appear to be relatively unaffected by peripheral axotomy.

Tice substrain BCG is used clinically as an immunotherapeutic agent against superficial bladder cancer. A boiling-water extract of this BCG showed anti-tumour activity against a murine S180 sarcoma model and was fractionated into three fractions, A, B and C, by the use of Sephadex LH-20 chromatography. An anti-tumour glucan, PS1A1, was isolated from fraction PS1A with Sephadex G-75. The molecular mass of PS1A1 was between 65 and 87 kDa by Sephadex G-100 chromatography. The structure of PS1A1 was investigated by one- and two-dimensional NMR spectroscopy and methylation analysis and was demonstrated to be primarily 1-->6-alpha-linked glucose units. We postulate that the repeating unit is: [Formula: see text]

Phospholipid-stabilized emulsion properties were studied in the presence of lysine, a water-structure breaker, using two unrelated procedures, photon correlation spectroscopy and a light obscuration instrument. Commercial Intralipid was used as a control. Lysine 0.125, 0.25 and 0.5 M induced changes in the size distribution of a non-heated model emulsion system, irrespective of any changes produced by environmental pH. Some of the laboratory-prepared emulsions containing lysine were more stable than the corresponding commercial heat-sterilized product Intralipid, once heated. The results suggest that lysine is producing an effect on the nascent oil-water interface that controls the physical stability of the system. Once the heat-induced interfacial rearrangement of the individual phospholipid molecules occurs, the influence of lysine becomes diminished.