Diagnostic application of two-color flow cytometry in 161 cases of hairy cell leukemia

Recent immunophenotypic studies of hairy cell leukemia (HCL) have suggested specific patterns of immunoreactivity that may aid in diagnosis. We studied peripheral blood (PB) from 161 cases of HCL using two-color direct immunofluorescence flow cytometry and an extended panel of antibody combinations. Circulating hairy cells were identified by immunophenotypic features in 92% of the cases and could be detected even when representing < or = 1% of circulating lymphocytes. The 133 cases with > or = 2% detectable hairy cells were analyzed in detail. HCL showed a uniform and unique B-cell phenotype, with each of the following features identified in 99% to 100% of cases: (1) positive staining for B-ly7, coexpressed with CD19; (2) very intense, uniform expression of CD11c, with CD19; (3) moderately intense staining for CD25, with CD19; (4) very intense staining for CD22; (5) moderate to very intense staining for CD20; and (6) moderately intense monoclonal surface Ig. Phenotypic variability existed in expression of CD10 (26%) and CD5 (4%). Based on these features, HCL was easily distinguished from 50 cases of chronic lymphocytic leukemia (CLL). Although CLL exhibited frequent expression of CD11c (74%) and CD25 (68%), the intensity of staining was significantly less than HCL. Furthermore, CLL was uniformly positive for CD5 and showed weak staining for CD20, CD22, and surface Ig. B-ly7 proved to be the most specific marker, reacting with 100% of HCL cases, but absent in all cases of CLL. We conclude that two-color flow cytometry with specific antibody combinations is an efficacious method for characterization and sensitive detection of hairy cells in PB. Application of the phenotypic criteria described should help to increase accuracy in diagnosis of HCL.     

Prediction of graft-versus-host disease by phenotypic analysis of early immune reconstitution after CD6-depleted allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic bone marrow transplantation (BMT). Because GVHD is frequently refractory to treatment, the early identification of high-risk patients could have significant clinical value. To identify such patients, we examined early immunologic recovery in 136 patients with hematologic malignancies who received anti-T12 (CD6)-purged allogeneic bone marrow over a 9-year period. The majority of patients received marrow from HLA-matched sibling donors after ablation with cyclophosphamide and total body irradiation. No patients received any immune suppressive medications for GVHD prophylaxis. The fraction and absolute numbers of peripheral blood lymphocytes (PBL) expressing the CD3, CD4, CD8, and CD56 surface antigens were determined weekly by immunofluorescence analysis in patients beginning 8 to 14 days (week 2) after marrow infusion. Results in patients who did or did not subsequently develop GVHD post-BMT were compared. Within 2 weeks of marrow infusion, patients who developed grades 2-4 GVHD had significantly higher percentages and absolute numbers of CD8+ T cells and a lower fraction of CD56+ natural killer (NK) cells than individuals who remained free of GVHD. Thirty-five percent of patients whose PBL were greater than 25% CD8+ in the second posttransplant week developed GVHD, compared with only 3% of patients who had < or = 25% CD8+ cells (odds ratio 37.8; 95% confidence interval [CI] 4.1 to 397). A subgroup of patients at very high risk for GVHD could be identified based on the combined frequency of CD8+ T cells and NK cells in blood. Seventy-five percent of patients with greater than 25% CD8+ cells and < or = 45% CD56+ cells during week 2 post-BMT developed GVHD, compared with only 11% of the remaining patients (odds ratio 24.9; 95% CI, 5.3 to 117.0). None of the 23 patients with both less than 25% CD8+ cells and greater than 45% CD56+ cells in the second posttransplant week developed grades 2-4 GVHD. Our findings indicate that CD8+ T cells play an important role in the pathogenesis of GVHD in humans. Analysis of immune reconstitution early after BMT is useful in predicting the onset of GVHD and can help direct the implementation of treatment strategies before the appearance of clinical manifestations. Such interventions may decrease the morbidity and mortality associated with allogeneic BMT and ultimately improve overall survival.     

A pilot study of extralevator abdominoperineal excision for primary melanoma of the anorectum

Melanoma of the anorectum represents <2 % of all anorectal cancers and 0.3 % of all primary melanomas. Prognosis is poor, and optimal surgical management is controversial. This series details the surgical management of patients with anorectal melanoma presenting between 2002 and 2013 to the Queen Elizabeth Hospital in Birmingham, UK, a tertiary referral centre for colorectal disease. A retrospective review of patient notes, histology reports, and clinical letters was used to assess perioperative course and long-term outcome of all surgical methods. Eight patients with a median age of 65.5 presented with primary anorectal melanoma during the study period. Six cases were staged as T4 pre-operatively, with two referred as incompletely excised polyps. All eight patients underwent abdominoperineal excision of the rectum (APER), with the most recent four cases undergoing extralevator APER. Clear resection margins were achieved in three out of the four patients in the extralevator APER group with no nodal spread detected at histological assessment. Extralevator APER appears to be feasible and safe in the treatment of melanoma of the anorectum, with 75 % medium-term survival (median 38 months) in selected patients. As it is known that clear margins at surgery are associated with disease-free survival, the wider excision margin at the level of the pelvic floor offered by extralevator APER could result in more favourable surgical outcomes in this prognostically poor malignancy.     

HL-T, a new cell line derived from HL-60 promyelocytic leukemia cell cultures expressing terminal transferase and secreting suppressor activity

A cell line with immature blast cell morphology was isolated from HL-60 promyelocytic leukemia cell cultures and designated HL-T. This new cell type is biphenotypic, expressing terminal transferase (TdT) together with myelomonocytoid immunologic features. TdT enzymatic activity, undetectable in HL-60, was determined to be 140 to 180 units/10(8) HL-T cells by the dGTP-assay, approximately 20% of the activity found in lymphoblastoid cell lines. HL-T predominantly synthesize the known 58- kDa TdT-protein plus a minor 54/56-kDa doublet. The 58-kDa steady state form is nonglycosylated and is phosphorylated. Precursor antigens S3.13 and MY-10, absent on HL-60, are expressed by HL-T; however, the cells are negative for HLA-Dr. Southern blot analysis by hybridization with immunoglobulin heavy chain (JH) and T cell-receptor chain gene (T beta) probes shows JH to be in the germ-line configuration in both cell lines and the T beta gene to be in germ-line in HL-60 but to be rearranged in HL-T. Truncation of the gene encoding the granulocyte-macrophage-colony- stimulating factor (GM-CSF), as found in HL-60, is not observed in HL- T. HL-T are resistant to differentiation-induction by retinoic acid and 1,25-dihydroxyvitamin D3. Cytogenetically HL-T share with HL-60 a deletion of the short arm of chromosome 9 at breakpoint p13, an aberration frequently found in patients with T cell leukemia. In addition, HL-T display t(8;9)(p11;p24) and trisomy 20. Tetraploidy is observed in 80% of HL-T metaphases with aberrations identical to those in the diploid karyotype. Like HL-60, the new line shows some surface- antigenic-T cell characteristics. Despite an antigenic pattern most consistent with that of helper-inducer T cells (T4+, D44+/-, 4B4+, 2H4- , TQ1+/-), HL-T cells and their conditioned culture medium suppress antigen, mitogen, and mixed-leukocyte-culture-mediated lymphocyte proliferation.     

Interleukin-2 production and response to interleukin-2 by peripheral blood mononuclear cells from patients after bone marrow transplantation: II. Patients receiving soybean lectin-separated and T cell-depleted bone marrow

The ability of peripheral blood mononuclear cells (PBMC) to produce and respond to interleukin-2 (IL-2) was evaluated in 50 recipients of HLA- identical bone marrow (BM) depleted of mature T cells by soybean agglutination and E rosetting (SBA-E-BM). In contrast to our previous findings in recipients of unfractionated marrow, during weeks 3 to 7 post-SBA-E-BM transplantation (BMT), PBMC from the majority of patients spontaneously released IL-2 into the culture medium. This IL-2 was not produced by Leu-11+ natural killer cells, which were found to be predominant in the circulation at this time, but by T11+, T3+, Ia antigen-bearing T cells. The IL-2 production could be enhanced by coculture with host PBMC frozen before transplant but not by stimulation with mitogenic amounts of OKT3 antibody, thus suggesting an in vivo activation of donor T cells or their precursors by host tissue. Spontaneous IL-2 production was inversely proportional to the number of circulating peripheral blood lymphocytes and ceased after 7 to 8 weeks post-SBA-E-BMT in most of the patients. In patients whose cells had ceased to produce IL-2 spontaneously or never produced this cytokine, neither coculture with host cells nor stimulation with OKT3 antibody thereafter induced IL-2 release through the first year posttransplant. Proliferative responses to exogenous IL-2 after stimulation with OKT3 antibody remained abnormal for up to 6 months post-SBA-E-BMT, unlike the responses of PBMC from recipients of conventional BM, which responded normally by 1 month post-BMT. However, the upregulation of IL- 2 receptor expression by exogenous IL-2 was found to be comparable to normal controls when tested as early as 3 weeks post-SBA-E-BMT. Therefore, the immunologic recovery of proliferative responses to IL-2 and the appearance of cells regulating in vivo activation of T cells appear to be more delayed in patients receiving T cell-depleted BMT. Similar to patients receiving conventional BMT, however, the ability to produce IL-2 after mitogenic stimulation remains depressed for up to 1 year after transplantation.     

Interleukin 2-activated killer cells in patients following transplants of soybean lectin-separated and E rosette-depleted bone marrow

During the early period following bone marrow transplantation before the immune system has reached full functional maturity, unprimed, nonspecific lytic systems may play a critical role as antiviral or antitumor effectors. The reconstitution of cells with this potential is of particular importance in recipients of bone marrow that has been depleted of mature T lymphocytes to prevent graft v host disease (GVHD). We examined the recovery of natural killer (NK) cells and interleukin 2 (IL 2)-augmented lymphokine-activated killer cells (LAK) in 48 patients at various intervals following transplantation of bone marrow depleted of mature cellular elements by treatment with soybean agglutinin and sheep RBCs (SBA-E- BMT). We found normal levels of both NK and LAK activity as early as 3 weeks following SBA-E- BMT. When compared with cells from controls, NK and LAK precursors from transplant recipients appeared to be activated in vivo in that freshly isolated peripheral blood mononuclear cells (PBMCs) from patients had an elevated cytolytic activity toward NK-insensitive targets and a more rapid response to activation by IL 2. In patients as well as controls, both LAK precursors and LAK effectors lacked antigens present on mature T lymphocytes (CD3, CD4, or CD8) but expressed antigens present on NK cells (CD2, CD16, and NKH1A). The LAK cells did not lyse either donor or host peripheral blood T cell targets. The activity of NK effectors but not LAK precursors survived the in vivo total body irradiation used for pretransplant conditioning in three patients studied. LAK precursors could be demonstrated as early as 18 days following transplant at a time when the bone marrow contained primarily donor- derived cells. Little or no LAK activity could be generated from cells of the SBA-E- BM graft itself, suggesting that LAK precursors differentiate rapidly from more primitive progenitors in the marrow graft. Thus, our data indicate that the NK and LAK lytic systems are among the earliest activities to recover during immune reconstitution following T cell-depleted BMTs.     

Hydrogen Peroxide Induced Deoxyribonucleic Acid Damage Preventive Activity of Selected <Emphasis Type="Italic">Valeriana</Emphasis> Species from West Himalaya

The polyphenols, antioxidant activity and deoxyribonucleic acid (DNA) damage preventive efficiency of 3 species Valeriana jatamansi, V. hardwickii and V. himalayana were investigated. V. himalayana exhibited significantly higher total phenolics and V. jatamansi showed significantly higher flavonoids and total tannins. Valerenic acid was significantly higher in V. himalayana (1.6 %) in root portion as compared to that of V. jatamansi (0.75 %). Significantly higher antioxidant activity of 2,2′-azinobis benzyl ethyl thiazole 6-sulphonic acid was observed as 4.65 mM AAE/100g in aerial portion and 5.73 mM AAE/100g in root portion; 2,2-diphenyl-1-picrylhydrzyl was 8.87 mM AAE/100g in aerial portion and 17.53 mM AAE/100g in root portion; and ferric reducing antioxidant power was 4.48 mM AAE/100g in aerial portion and 7.28 mM AAE/100g in root portion was higher in V. himalayana as compared to that of V. jatamansi and V. hardwickii. DNA damage preventive efficiency revealed variation in these species. V. himalayana exhibited better ability to prevent Fenton reagent induced DNA damage (97.97 %) as compared to the other two species at 100 μg/μl of aqueous extract. Based on the results it is recommended that as V. himalayana exhibited higher phytochemicals, antioxidant property and DNA damage preventive efficiency, therefore, more systematic investigation and conservation of this species is suggested to meet the increasing industrial demand. As the species is threatened in its natural habitat the other two species i.e., V. hardwickii and V. jatamansi can be promoted as an alternative source for phenolics and antioxidants.     

Transethosomes: Cutting edge approach for drug permeation enhancement in transdermal drug delivery system

The skin is a major route of drug administration. Despite the high surface area of the skin, drug delivery via the skin route is problematic due to its physiological obstacles. The formulation scientist has developed a vesicular system to enhance the skin's absorption of bioactive substances. Among numerous vesicular systems, concept of transethosomes (TEs) introduced in 2012 are being tested for drug delivery to the dermis. When transferosomes and ethosomes interact, TEs are produced. It consists of water, ethanol, phospholipids, and an edge activator. Ethanol and the edge activator increase the absorption of medication through the skin. In the presence of ethanol and an edge activator, skin permeability can increase. The advantages of TEs include increased patient compliance, bypassing first-pass metabolism, including non-toxic raw components, being a noninvasive method of drug delivery, being more stable, biocompatible, biodegradable, and administered in semisolid form. TEs can be produced through the use of hot, cold, mechanical dispersion, and conventional techniques. The morphology, shape, size, zeta potential, drug loading efficiency, vesicle yield, biophysical interactions, and stability of TEs define them. Recent studies reported successful transdermal distribution of antifungal, antiviral, anti-inflammatory, and cardiovascular bioactive while using ethosomes with significant deeper penetration in skin. The review extensively discussed various claims on TEs developed by researchers, patents, and marketed ethosomes. However, till today no patens being granted on TEs. There are still lingering difficulties related to ethanol-based TEs that require substantial research to fix.     

Multitargeted C9-substituted ester and ether derivatives of berberrubine for Alzheimer's disease: Design,synthesis, biological evaluation,metabolic stability,and pharmacokinetics

Berberrubine is a naturally occurring isoquinoline alkaloid and a bioactive metabolite of berberine. Berberine exhibits a wide range of pharmacological activities, including cholinesterase inhibition. The cholinesterase inhibitors provide symptomatic treatment for Alzheimer's disease; however, multitarget-directed ligands have the potential as disease-modifying therapeutics. Herein, we prepared a series of C9-substituted berberrubine derivatives intending to discover dual cholinesterase and beta-site amyloid-precursor protein cleaving enzyme 1 (BACE-1) inhibitors. Most synthesized derivatives possessed balanced dual inhibition (AChE and BChE) activity in the submicromolar range and a moderate inhibition against BACE-1. Two most active ester derivatives, 12a and 11d , display inhibition of AChE, BChE, and BACE-1. The 3-methoxybenzoyl ester derivative, 12a , inhibits electric eel acetylcholinesterase (EeAChE), equine serum butyrylcholinesterase (eqBChE), and human hBACE-1 with IC₅₀ values of 0.5, 4.3, and 11.9 μM, respectively and excellent BBB permeability (P_e = 8 × 10⁻⁶ cm/s). The ester derivative 12a is metabolically unstable; however, its ether analog 13 is stable in HLM and exhibits inhibition of AChE, BChE, and BACE-1 with IC₅₀ values of 0.44, 3.8, and 17.9 μM, respectively. The ether analog also inhibits self-aggregation of Aβ and crosses BBB (P_e = 7.3 × 10⁻⁶ cm/s). Administration of 13 at 5 mg/kg (iv) in Wistar rats showed excellent plasma exposure with AUC_0−∞ of 28,834 ng min/ml. In conclusion, the multitargeted berberrubine ether derivative 13 is CNS permeable and has good ADME properties.