Modified Donor Lymphocyte Infusion (DLI) for the Prophylaxis of Leukemia Relapse after Hematopoietic Stem Cell Transplantation in Patients with Advanced Leukemia—Feasibility and Safety Study

PURPOSE: We retrospectively evaluated the feasibility and safety of a modified prophylactic donor lymphocytes infusion (DLI) approach in advanced leukemia. MATERIALS AND METHODS: Thirty-three patients with advanced leukemia received modified prophylactic DLI; that is, granulocyte colony-stimulating factor-primed peripheral blood progenitor cells instead of steady-donor lymphocyte harvests were used, and a short-term immunosuppressive agent (cyclosporine A or methotrexate 10 mg once per week for 2 to 4 weeks) was used for prevention of DLI-associated graft versus host disease (GVHD) after human leukocyte antigen-identical sibling hematopoietic stem cell transplantation. RESULTS: Thirty-nine infusions were performed in 33 patients. The mononuclear cells and median CD3+ cells infused for DLI were 1-2 x 10(8) and 0.93 x 10(6) per kilogram, respectively. Six patients experienced II-IV-grade acute GVHD. Twenty patients developed chronic GVHD. No GVHD-related death or transfusion-related pancytopenia was observed. With an 18-month median follow-up, 16 patients were in disease-free survival, and overall survival at 1 and 1.5 years was 69.0% and 50.2%, respectively. CONCLUSION: The modified prophylactic DLI strategy might represent a step forward in the treatment of advanced leukemia.     

��-Site APP-cleaving enzyme 1 (BACE1) cleaves cerebellar Na+ channel ��4-subunit and promotes Purkinje cell firing by slowing the decay of resurgent Na+ current

In cerebellar Purkinje cells, the β4-subunit of voltage-dependent Na(+) channels has been proposed to serve as an open-channel blocker giving rise to a "resurgent" Na(+) current (I (NaR)) upon membrane repolarization. Notably, the β4-subunit was recently identified as a novel substrate of the β-secretase, BACE1, a key enzyme of the amyloidogenic pathway in Alzheimer's disease. Here, we asked whether BACE1-mediated cleavage of β4-subunit has an impact on I (NaR) and, consequently, on the firing properties of Purkinje cells. In cerebellar tissue of BACE1-/- mice, mRNA levels of Na(+) channel α-subunits 1.1, 1.2, and 1.6 and of β-subunits 1-4 remained unchanged, but processing of β4 peptide was profoundly altered. Patch-clamp recordings from acutely isolated Purkinje cells of BACE1-/- and WT mice did not reveal any differences in steady-state properties and in current densities of transient, persistent, and resurgent Na(+) currents. However, I (NaR) was found to decay significantly faster in BACE1-deficient Purkinje cells than in WT cells. In modeling studies, the altered time course of I (NaR) decay could be replicated when we decreased the efficiency of open-channel block. In current-clamp recordings, BACE1-/- Purkinje cells displayed lower spontaneous firing rate than normal cells. Computer simulations supported the hypothesis that the accelerated decay kinetics of I (NaR) are responsible for the slower firing rate. Our study elucidates a novel function of BACE1 in the regulation of neuronal excitability that serves to tune the firing pattern of Purkinje cells and presumably other neurons endowed with I (NaR).     

Detection of bacillus Galmette-Guérin (Mycobacterium bovis BCG) DNA in urine and blood specimens after intravesical immunotherapy for bladder carcinoma

A real-time PCR targeting IS6110 was employed for the detection of Mycobacterium tuberculosis DNA in specimens collected from 10 patients treated with intravesical M. bovis bacillus Galmette-Guérin (BCG) immunotherapy for bladder malignancy. BCG DNA was detected in all urine specimens taken 24 h after the instillations, as well as in 24% of the specimens collected 7 days after the instillations; it was also detected in a single specimen taken 6 weeks after the last instillation. BCG DNA was detected in 8.3% of the blood specimens taken 1 day after instillation, and its amplification was associated with cases of self-limiting fever. These findings give indications that this real-time PCR is helpful to recognize BCG bacteremic cases, which may lead to mycobacterial infection.     

Control of Type I Collagen Systhesis: Evidence for Pretranslational Coordination of Proα1 (I) and Proα2 (I) Chain Synthesis in Embryonic Chick Bone

The normal type I collagen molecule contains two αl (I) chains and one α2 (I) chain. In embryonic chick calvaria, the two chains are synthesized in a 2: 1 ratio, and total polysomes from this tissue contain twice as much mRNA for proal (I) as for proα2 (I).⁹ To further investigate the mechanism by which synthesis may be coordinated, RNA isolated from various cell fractions of embryonic chick calvaria was translated in a rabbit reticulocyte lysate cell-free system. The procollagen chain products were separated by gel-electrophoresis and densitometrically quantitated from autoradiograms of the gels. Total cellular RNA, total cytoplasmic RNA, and polysomal RNA each directed the synthesis of proal (I) and proa2 (I) in a proportion of 2: 1, whereas no procollagen mRNA activity was found in nonpolysomal cytoplasmic RNA. These results indicate that in the chick bone cells, all compartments contain twice as much proal (I) mRNA as proa2 (I) mRNA, and that virtually all procollagen mRNA in the cytoplasm is poly some-bound. The coordination of procollagen chain synthesis thus presumably occurs at a pretranslational level, through differential rates of formation and/or degradation of the two mRNAs.     

Japanese monkey (<Emphasis Type="Italic">Macaca fuscata</Emphasis>) with alveolar echinococcosis after treatment with albendazole for 10 years: serodiagnosis and determination of albendazole metabolites

In 1997, an outbreak of alveolar echinococcosis in Japanese monkeys (Macaca fuscata) occurred in a zoo in Hokkaido, Japan. Twelve infected monkeys from a colony (n = 57) were diagnosed serologically by Western blotting, and ultrasonography showed the presence of tumor-like tissue in the livers of nine monkeys. The 12 infected monkeys have been treated with albendazole for 10 years without surgical resection. Ten of these monkeys have died so far; diagnoses were confirmed histopathologically through autopsy. Two of these monkeys are still alive. Recently, a significant difference between the two living monkeys was recognized. A difference in curative effect was demonstrated between the two living monkeys by radiography, contrast enhanced computed tomography, and contrast ultrasound. One showed metastasis to various organs, and the other appeared to be almost cured, as demonstrated by size reduction and calcification of the lesion after albendazole treatment for 10 years. This time, serological reexamination was performed to corroborate this apparent difference. The serological tests supported the preliminary imaging findings. In addition, the presence of albendazole metabolites in sera was confirmed by high-performance liquid chromatography. In this study, it was demonstrated that tests which have been used in human cases were also effective for diagnosing alveolar echinococcosis and for assessing curative effects in nonhuman primates such as M. fuscata.     

Engineering poly(lactic-<Emphasis Type="Italic">co</Emphasis>-glycolic acid) (PLGA) micro- and nano-carriers for Controlled Delivery of 17β-Estradiol

With menopause, circulating levels of 17β-estradiol (E2) markedly decrease. E2-based hormone therapy is prescribed to alleviate symptoms associated with menopause. E2 is also recognized for its beneficial effects in the central nervous system (CNS), such as enhanced cognitive function following abrupt hormonal loss associated with ovariectomy. For women with an intact uterus, an opposing progestogen component is required to decrease the risk of developing endometrial hyperplasia. While adding an opposing progestogen attenuates these detrimental effects on the uterus, it can attenuate the beneficial effects of E2 in the CNS. Poly(lactic-co-glycolic acid) (PLGA) micro- and nano- carriers (MNCs) have been heavily investigated for their ability to enhance the therapeutic activity of hydrophobic agents following exogenous administration, including E2. Multiple PLGA MNC formulation parameters, such as composition, molecular weight, and type of solvent used, can be altered to systematically manipulate the pharmacokinetic and pharmacodynamic profiles of encapsulated agents. Thus, there is an opportunity to enhance the therapeutic activity of E2 in the CNS through controlled delivery from PLGA MNCs. The aim of this review is to consider the fate of exogenously administered E2 and discuss how PLGA MNCs and route of administration can be used as strategies for controlled E2 delivery.     

Expressions of Somatostatin Receptor Subtypes (SSTR-1, 2, 3, 4 and 5) in?Neuroblastic Tumors; Special Reference to Clinicopathological Correlations with?International Neuroblastoma Pathology Classification and Outcomes

Somatostatin receptor (SSTR) expressions in neuroblastomas (NBs) have been confirmed employing various methods. High SSTR-2 expression was suggested to be a favorable prognostic marker, though little is known about the relationships between the expressions of SSTR subtypes, other than SSTR-2, and prognosis. We investigated the expressions of all five known SSTR subtypes in 63 neuroblastic tumors (NTs), employing immunohistochemistry, and also conducted quantitative real-time RT-PCR in 37 of these tumors. We evaluated correlations between the expressions of SSTR subtypes and prognosis, based on the International Neuroblastoma Pathology Classification and patient outcomes. More than 90% of cases expressed, at a minimum, SSTR-1 and/or 2. Ganglioneuromas and ganglioneuroblastomas expressed more than two SSTR subtypes. Among NBs, the favorable histology group showed higher SSTR subtype expressions than the unfavorable histology group. The same tendency was observed when surviving and deceased cases were compared, though SSTR-2 expression was well preserved in some of the deceased cases. In conclusion, NTs highly expressed SSTR-1 and/or 2, and expressions of SSTR generally indicate a good prognosis. However, even those in the unfavorable histology group with NBs expressing SSTR are good candidates for molecular targeting therapy using somatostatin analogues.     

Recurring multifocal leiomyosarcoma of the urinary bladder 22 years after therapy for bilateral (hereditary) retinoblastoma: A case report and review of the literature

We report on a case of urinary bladder leiomyosarcoma in a 23-year-old woman, 22 years after therapy for bilateral retinoblastoma. The tumor presented with dysuria and macroscopic haematuria. Cystoscopy revealed a tumor localized in the trigonum covered by an ulcerated urothelium. The patient underwent a transvesical tumor resection. Eight months later, a second leiomyosarcoma developed in the vertex, at a site different from the previous one. A cystoscopic trans-urethral tumor resection was performed, followed by combined chemotherapy. One year later another recurrence occurred at the site of the primary resection. Open laparotomic resection of the involved bladder wall was performed. The patient remains both recurrence and metastases free after twenty months of follow-up. Molecular analysis of the peripheral blood showed rare germline point mutation in the intron 24 of the RB1 gene. FISH analysis of the tumor tissue revealed polyploid cells with relative loss of normal RB1 gene locus, indicating deletion and second hit loss of the second RB1 allele function. Along with the ten previously reported cases, this report suggests a non-random association between the hereditary retinoblastoma and urinary bladder leiomyosarcoma. Therapy with cyclophosphamide seems to be an important risk factor. Life-long surveillance for second malignancies, including bladder leiomyosarcoma is therefore mandatory in these patients. Keywords: retinoblastoma - urinary bladder - leiomyosarcoma - secondary cancer - cyclophosphamide.     

Genes for C4b-binding protein α- and β-chains (C4BPA andC4BPB) are located on chromosome 1, band 1q32, in humans and on chromosome 13 in rats

C4b-binding protein is involved in the regulation of the complement system. It is a multimeric protein composed of seven identical -chains and a single copy of a unique -chain. The latter was identified only recently and its structure determined by cDNA cloning. Both subunits in C4b-binding protein belong to the same superfamily of proteins composed predominantly of tandemly arranged short consensus repeats (SCR) approximately 60 amino acid residues in length. The gene for the human -chain is known to be located in a gene cluster on chromosome 1, band 1q32, which is called the regulators of complement activation (RCA) gene cluster. We have used cDNA probes for both - and -chains of human C4b-binding protein to localize their genes with an in situ hybridization technique. We find the genes for both chains to be located on chromosome 1, band 1q32, in the human. This suggests that the -chain gene is also a member of the RCA gene cluster and that the - and -chain genes are located close to each other. The cDNA probes for the - and -chains also were used to screen mouse-rat somatic cell hybrids using Southern blotting to localize their genes in the rat. Both the - and -chain genes were shown to be located on chromosome 13 in the rat. These are the second and third genes to be located on rat chromosome 13, and the results suggest that the genes for the - and the -chains together with the gene for coagulation factor V represent a conserved chromosomal region in rat and man.     

Screening for antifeedant and larvicidal activity of plant extracts against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Hübner), <Emphasis Type="Italic">Sylepta derogata</Emphasis> (F.) and <Emphasis Type="Italic">Anopheles stephensi</Emphasis> (Liston)

Plant extracts, especially botanical insecticides, are currently studied more and more because of the possibility of their use in plant protection. Biological activity of five solvent plant extracts were studied using fourth instar larvae of gram pod borer Helicoverpa armigera (Lepidoptera: Noctuidae), cotton leaf roller Sylepta derogata (Lepidoptera: Pyralidae) and malaria vector Anopheles stephensi (Diptera: Culicidae). Antifeedant and larvicidal activity of acetone, chloroform, ethyl acetate, hexane and methanol peel, leaf and flower extracts of Citrus sinensis, Ocimum canum, Ocimum sanctum and Rhinacanthus nasutus were used in this study. During preliminary screening, the extracts were tested at 1,000 ppm concentration. The larval mortality was observed after 24 h of exposure. All extracts showed moderate larvicidal effects; however, the highest larval mortality was found in peel chloroform extract of C. sinensis, flower methanol extract of O. canum against the larvae of H. armigera (LC₅₀ = 65.10,51.78, LC₉₀ = 277.39 and 218.18 ppm), peel methanol extract of C. sinensis, flower ethyl acetate extract of O. canum and leaf acetone extract of O. sanctum against the larvae of S. derogata (LC₅₀ = 20.27,58.21,36.66, LC₉₀ =113.15,285.70 and 668.02 ppm), peel methanol extract of C. sinensis, leaf and flower ethyl acetate extracts of O. canum against the larvae of A. stephensi (LC₅₀ = 95.74,101.53,28.96, LC₉₀ = 303.20,492.43 and 168.05 ppm), respectively. These results suggest that the chloroform and methanol extract of C. sinensis, ethyl acetate flower extracts of O. canum and acetone extract of O. sanctum have the potential to be used as an ideal eco-friendly approach for the control of the agricultural pests H. armigera, S. derogata and medically important vector A. stephensi.     

Comprehensive immunohistochemical analysis of Her-2/neu oncoprotein overexpression in breast cancer: HercepTest™ (Dako) for manual testing and Her-2/neuTest 4B5 (Ventana) for Ventana BenchMark automatic staining system with correlation to results of fluorescence in situ hybridization (FISH)

Overexpression of Her-2/neu-oncoprotein is used as marker for Herceptin® therapy. To investigate the sensitivity and specificity of automatic immunohistochemistry (Benchmark, Ventana), we compared the results to the manual testing (Dako) in 130 breast carcinomas and validated the results by fluorescence in situ hybridization (FISH). Manual and automatic immunohistochemistry of Her-2/neu-oncoprotein using two different antibodies (HercepTest?, Her-2/neuTest 4B5) was analyzed. FISH was performed in all cases with uncertain or strong overexpression in either immunohistochemical stainings or with different immunohistochemical results. Same immunohistochemical results were seen in 73.8%. Two cases with overexpression, detected with Her-2/neuTest 4B5 and confirmed by FISH, showed no overexpression using HercepTest?. From 21 cases with 2+ by Her-2/neuTest 4B5, 15 cases had no gene amplification (two of them with 3+ HercepTest?); three cases showed a gene amplification (one of them with failing overexpression by HercepTest?); two other cases were polysomic; one could not be analyzed. Ventana immunohistochemistry seems to be of same reliability like Dako with a little better concordance to FISH in our study.     

Occurrence of ocular melanoma thirteen years after skin melanoma: two separate primaries or metastatic disease? A case solved with <Emphasis Type="Italic">NRAS</Emphasis> and <Emphasis Type="Italic">CDKN2A (INK4A-ARF)</Emphasis> mutational analysis

The differential diagnosis between primary uveal melanoma and cutaneous melanoma metastasis in the eye may be difficult, both clinically and histologically. We report successful application of combined mutational analysis of the NRAS and the CDKN2A gene to discriminate between these two entities. The patient had a history of a superficial spreading cutaneous melanoma of the left shoulder. Nine years later, she developed a lymph node metastasis in the left axilla, and 13 years later she presented with an atypical, pigmented tumor in the uvea. Histologically, the origin of the uveal melanoma could not be determined with certainty. We performed molecular analysis on the skin melanoma, the lymph node metastasis and the uveal melanoma. We detected an NRAS codon 61 mutation (c.182A>G, p.Gln61Arg) in all three tumor specimens. This mutation was absent in the normal control tissue of the patient, thereby excluding a germline mutation. To confirm a clonal relationship between the tumors, we also performed CDKN2A mutational analysis. We detected a CDKN2A mutation ((p16) c.238C>T, p.Arg80X, (p14) c.404C>T, p.Pro135Leu)) in the tumor samples, but not in the normal control tissue of the patient. We concluded that the uveal melanoma is a metastasis from the cutaneous melanoma removed 13 years before.     

MRSA detection: comparison of two molecular methods (BD GeneOhm<Superscript>®</Superscript> PCR assay and <Emphasis Type="Italic">Easy</Emphasis>-Plex) with two selective MRSA agars (MRSA-ID and Oxoid MRSA) for nasal swabs

Screening of patients is an important component for methicillin-resistant Staphylococcus aureus (MRSA) containment. The aim of this study was to determine the utility of molecular methods compared to selective agars for MRSA detection. Consecutive high risk patients (n = 200) were screened for nasal MRSA colonization using chromogenic selective agars (MRSA ID and CMRSA) as well as the BD GeneOhm MRSA (previously known as IDI-MRSA) and the Easy-Plex commercial real time PCR assays. The sensitivities of the molecular methods were similar to the BD GeneOhm MRSA (86%) and Easy-Plex (84%). The chromogenic agars, MRSA ID and CMRSA, showed sensitivities of 62% and 84%, respectively. Sensitivity was influenced by both the MRSA antibiograms and MRSA clonal type. The specificity was >98% in all methods except for the Easy-Plex assay (90%). Sensitivity of selective agars increased with 48 hours of incubation with a corresponding decrease in specificity. In conclusion, molecular detection methods for MRSA remain sensitive and rapid but are associated with greater expense.     

Should we expand the indications for the DAIR (debridement,antibiotic therapy,and implant retention) procedure for <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> prosthetic joint infections? A multicenter retrospective study

To evaluate factors associated with failure in patients treated with DAIR (debridement, antibiotic therapy, and implant retention) for Staphylococcus aureus prosthetic joint infections (PJIs). We retrospectively analyzed consecutive patients with stable PJI due to S. aureus treated with DAIR at six hospitals between 2010 and 2014. Cox proportional hazards regression was used to study factors associated with treatment failure at 2 years. Of 154 eligible patients, 137 were included (mean age 73?±?13 years; male 56%). The estimated success rate according to the Kaplan–Meier method was 76.2 [95% CI 68–83] at 2 years of follow-up. In multivariate analysis, longer duration of treatment (hazard ratio (HR) 0.78 [0.69–0.88]; p?<?0.001) and combination therapy including rifampin (HR 0.08 [0.018–0.36]; p?=?0.001) were independently associated with success, whereas active smoking was independently associated with failure (HR 3.6 [1.09–11.84]; p?=?0.036). When the analysis was restricted to patients with early infection onset (<?3 months), early acute infection was also predictive of a better prognosis (HR 0.25 [0.09–0.7]; p?=?0.009). Failure was not associated with time from prosthesis insertion to debridement, nor with duration of symptoms >?3 weeks and type of prosthesis (hip or knee). These results remained unchanged when the 14 patients under immunosuppressive therapy were removed from analysis. These data suggest that DAIR can be performed even if infection and symptoms are delayed but reserved to patients who are able to follow rifampin-based combination therapy for a prolonged duration that should not be different for hip and knee PJI.     

Utility of ITS1–5.8S–ITS2 sequences for species discrimination and phylogenetic inference of two closely related bucephalid digeneans (Digenea: Bucephalidae): <Emphasis Type="Italic">Dollfustrema vaneyi</Emphasis> and <Emphasis Type="Italic">Dollfustrema hefeiensis</Emphasis>

The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from 60 specimens belonging to two closely related bucephalid digeneans (Dollfustrema vaneyi and Dollfustrema hefeiensis) from different localities, hosts, and microhabitat sites were cloned to examine the level of sequence variation and the taxonomic levels to show utility in species identification and phylogeny estimation. Our data show that these molecular markers can help to discriminate the two species, which are morphologically very close and difficult to separate by classical methods. We found 21 haplotypes defined by 44 polymorphic positions in 38 individuals of D. vaneyi, and 16 haplotypes defined by 43 polymorphic positions in 22 individuals of D. hefeiensis. There is no shared haplotypes between the two species. Haplotype rather than nucleotide diversity is similar between the two species. Phylogenetic analyses reveal two robustly supported clades, one corresponding to D. vaneyi and the other corresponding to D. hefeiensis. However, the population structures between the two species seem to be incongruent and show no geographic and host-specific structure among them, further indicating that the two species may have had a more complex evolutionary history than expected.