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Abstract Background Rapid diagnostic tests (RDTs) have revolutionized the diagnosis of malaria. Among the various factors affecting RDTs sensitivity is genetic variation of the antigen used. The genetic variation in PfHRP2 and PfHRP3 proteins was studied among the Indian Plasmodium falciparum isolates. Methods One hundred and forty isolates of P. falciparum were collected from six geographical regions of India. Target genes encoding PfHRP2 and PfHRP3 antigens were sequenced to study genetic polymorphism. Minimum detection limit giving a positive rapid diagnostic test was also determined. Results Extensive variations were observed in amino acid repeat types of PfHRP2 and PfHRP3. PfHRP2 exhibited more polymorphism than PfHRP3. Significant relation was observed between type 2 and type 7 repeats and RDT detection rate as higher number of these repeats showed better sensitivity with RDTs. Conclusion The results provide insights into the genetic diversity of Pfhrp2 and Pfhrp3 genes among Indian P. falciparum population and its relation to RDT sensitivity.

Abstract Background Resistance in Plasmodium falciparum to commonly used anti-malarial drugs, especially chloroquine, is being increasingly documented in India. By 2007, the first-line treatment for uncomplicated malaria has been revised to recommend artemisinin-based combination therapy (ACT) for all confirmed P. falciparum cases. Objective The objective of this study was to compare the efficacy, safety and tolerability between dihydroartemisinin-piperaquine (DP) and artesunate plus mefloquine (A + M) drug combinations in the treatment of uncomplicated P. falciparum malaria in India. Methods Between 2006 and 2007, 150 patients with acute uncomplicated P. falciparum malaria were enrolled, randomized to DP (101) or A + M (49) and followed up for 63 days as part of an open-label, non-inferiority, randomized, phase III multicenter trial in Asia. Results The heterogeneity analysis showed no statistically significant difference between India and the other countries involved in the phase III study, for both the PCR-corrected and uncorrected cure rates. As shown at the whole study level, both forms of ACT were highly efficacious in India. In fact, in the per protocol population, the 63-day cure rates were 100% for A + M and 98.8% for DP. The DP combination exerted a significant post-treatment prophylactic effect, and compared with A + M a significant reduction in the incidence of new infections for DP was observed (respectively 17.1% versus 7.5% of patients experienced new infection within follow up). Parasite and fever clearance was rapid in both treatment arms (median time to parasite clearance of one day for both groups). Both DP and A + M were well tolerated, with the majority of adverse events of mild or moderate severity. The frequencies of individual adverse events were generally similar between treatments, although the incidence of post treatment adverse events was slightly higher in patients who received A + M with respect to those treated with DP. Conclusion DP is a new ACT displaying high efficacy and safety in the treatment of uncomplicated P. falciparum malaria and could potentially be considered for the first-line treatment of uncomplicated falciparum malaria in India. Trial registration Current Controlled Trials ISRCTN 81306618

Abstract Background Artemisinin-based combination therapy (ACT) has been recommended for the treatment of falciparum malaria by the World Health Organization. Though India has already switched to ACT for treating falciparum malaria, there is need to have multiple options of alternative forms of ACT. A randomized trial was conducted to assess the safety and efficacy of the fixed dose combination of artesunate-amodiaquine (ASAQ) and amodiaquine (AQ) for the treatment of uncomplicated falciparum malaria for the first time in India. The study sites are located in malaria-endemic, chloroquine-resistant areas. Methods This was an open label, randomized trial conducted at two sites in India from January 2007 to January 2008. Patients between six months and 60 years of age having Plasmodium falciparum mono-infection were randomly allocated to ASAQ and AQ arms. The primary endpoint was 28-day PCR-corrected parasitological cure rate. Results Three hundred patients were enrolled at two participating centres, Ranchi, Jharkhand and Rourkela, Odisha. Two patients in AQ arm had early treatment failure while there was no early treatment failure in ASAQ arm. Late treatment failures were seen in 13 and 12 patients in ASAQ and AQ arms, respectively. The PCR-corrected cure rates in intent-to-treat population were 97.51% (94.6-99.1%) in ASAQ and 88.65% (81.3-93.9%) in AQ arms. In per-protocol population, they were 97.47% (94.2-99.2%) and 88.30% (80-94%) in ASAQ and AQ arms respectively. Seven serious adverse events (SAEs) were reported in five patients, of which two were reported as related to the treatment. All SAEs resolved without sequel. Conclusion The fixed dose combination of ASAQ was found to be efficacious and safe treatment for P. falciparum malaria. Amodiaquine also showed acceptable efficacy, making it a suitable partner of artesunate. The combination could prove to be a viable option in case India opts for fixed dose combination ACT. Clinical trial registry ISRCTN84408319

We performed aCGH, SKY/FISH, molecular mapping and expression analyses on a permanent CD8+ NK/T cell line, 'SRIK-NKL' established from a lymphoma (ALL) patient, in attempt to define the fundamental genetic profile of its unique NK phenotypes. aCGH revealed hemizygous deletion of 6p containing genes responsible for hematopoietic functions. The SKY demonstrated that a constitutive reciprocal translocation, rcpt(5;14)(p13.2;q11) is a stable marker. Using somatic hybrids containing der(5) derived from SRIK-NKL, we found that the breakpoint in one homologue of no. 5 is located upstream of IL7R and also that the breakpoint in no. 14 is located within TRA@. The FISH analysis using a BAC which contains TRA@ and its flanking region further revealed a approximately 231kb deletion within 14q11 in the der(5) but not in the normal homologue of no. 14. The RT-PCR analysis detected mRNA for TRA@ transcripts which were extending across, but not including, the deleted region. IL7R was detected at least at mRNA levels. These findings were consistent with the immunological findings that TRA@ and IL7R are both expressed at mRNA levels and TRA@ at cytoplasmic protein levels in SRIK-NKL cells. In addition to rcpt(5;14), aCGH identified novel copy number abnormalities suggesting that the unique phenotype of the SRIK-NKL cell line is not solely due to the TRA@ rearrangement. These findings provide supportive evidence for the notion that SRIK-NKL cells may be useful for studying not only the function of NK cells but also genetic deregulations associated with leukemiogenesis.

Progesterone (P), required for successful pregnancy, influences autoimmune, infectious, and malignant diseases via adaptive and innate immune effects. P induces NK inhibitor progesterone induced blocking factor (PIBF) in CD8+ T cells. PIBF isoforms could permit solid tumor immune escape. Expression and modulation of PIBF and innate immune proteins by P in leukemia cells and leukocyte subpopulations have not been reported. Ten T, seven myeloid, six B, five epithelial, fibroblast BG9, G-CSF mobilized CD34+ stem cells, and peripheral blood mononuclear cells were screened for PIBF mRNA by RT-PCR, and protein by immunohistochemistry in SRIK-NKL, MOT, U937, HL60, R-CLL, MD-E, 729pH6neo, SRIH-B(ATL), SRIK-B(T-PLL), and MeWo. Cell lines expressing PIBF and exemplifying myeloid/monoblast, natural killer/T, and B lineages were cultured with and without 0.5 - 5 microM P or 0.5 - 0.05 microM mifepristone (RU486) for 24 h. Subsequently they were examined for changes in the expression of mRNA by RT-PCR and protein by immunohistochemistry for PIBF and some innate immune factors. All cells expressed PIBF mRNA; protein only in four (SRIK-NKL, U937, SRIK-B(T-PLL) and HL60) out of 10 cell lines tested. P increased and RU486 decreased PIBF in U937, SRIK-B(T-PLL) and SRIK-NKL. P upregulated TLR-4 in U937, and HNP1 - 3, LL-37, IRAK-2, and IRAK-4 in multiple lines and RU486 down regulated these. PIBF may be used by some leukemias to evade immune surveillance and is a potential therapeutic target. P may impact infection and autoimmunity via effects on LPS receptor, TLR signaling, and antimicrobial peptides.

T-cells and NK cells arise from common pluripotent stem cells, with shared early developmental pathway and surface markers. Distinguishing between them is becoming difficult, but critical for study. A large family of NK cells, including classical NK, NK-T, and NK-CTL exists. Natural cytotoxicity receptors (NKp46, NKp44, NKp30) have been proposed as specific for classical NK cells, but were expressed at protein and mRNA level by CD8+NK/T cell line SRIK-NKL, suggesting more widespread expression. We investigated and found expression of these markers at the protein and mRNA level in multiple human cell lines.

Natural killer (NK) cells, innate lymphocyte effectors, kill virus-infected host and tumor cells in non-MHC, non-TCR restricted fashion, unlike T cells. The role of NK cells in recognition and killing of pathogens remains unknown. Expression of the ten human pathogen associated molecular pattern or toll-like receptors (TLR's), associated molecules, and antimicrobial peptides alpha, beta defensins, and LL-37 by NK cells has not been investigated previously. We report our CD8+ NK/T cell line SRIK-NKL, derived from leukemic phase of acute lymphoblastic lymphoma, expresses mRNA and protein for 8/10 TLR's, associated proteins for signaling, defensins, cathelicidin/LL-37, and responds to live bacteria by cell proliferation and increased IFN-gamma, TNF-alpha, TNF-beta, and MIP-1alpha production.

The distinction between T cells and NK cells is difficult, and becoming more complex, as the diversity of the human lymphocyte repertoire is evident. We report the establishment of a permanent CD8+ NK/T cell line (SRIK-NKL) from a patient with leukemic phase of acute lymphoblastic lymphoma having characteristics of both NK and T cells, and extensively describe its phenotype, including cytotoxic activity, NK cell receptor expression, and other molecules critical for immune function. We further compare SRIK-NKL to other available NK/NK-T cell lines. SRIK-NKL may be useful for studying NK cell development, functions, and modulation, leading to novel strategies for treatment of autoimmune disease, infection, and cancer.

Problem: Despite evidence indicating a role for hepatocyte growth factor (HGF) in gastrointestinal and reproductive physiology, the concentration and distribution of HGF in human breast milk (BM) and reproductive tract fluids remain unknown., Method of Study: Using enzyme-linked immunosorbent assay (ELISA), the HGF concentrations were determined in human oviductal fluid (hOF), follicular fluid (FF), amniotic fluid (AF), seminal plasma (SP), and colostrum/milk samples, and expression of HGF mRNA by milk cells and AF cells were examined by reverse transcriptase-polymerase chain reaction (RT-PCR)., Results: HGF is present at nearly 70-fold normal serum (0.85+/-0.15 ng/mL) concentration in FF (n = 3; x = 57+/-16 ng/mL) and AF (n = 17; x = 57+/-26 ng/mL), and is also present in hOF (n = 3; x = 4.8+/-2.3 ng/mL) and CVL (n = 8; x = 0.7+/-1.1 ng/mL) varying throughout the menstrual cycle. HGF is found at 3-times serum concentration in BM (n = 24; x = 2.3+/-1.3 ng/mL) with no significant difference between premature and full term or stage of lactation (colostrum, transitional, mature milk). HGF mRNA was detected in BM cells but not in AF cells., Conclusions: HGF is present in sufficient amounts to profoundly affect gastrointestinal maturation in the fetus via swallowed AF and neonate via BM, and helps to explain the increased rate of necrotizing enterocolitis (NEC) in infants of premature rupture of membrane (PROM)-complicated pregnancies, and the decreased rate in breast fed neonates. HGF in FF may be necessary for the development and maturation of the oocyte. HGF in hOF, SP, and cervicovaginal lavage (CVL) is likely to enhances epithelial cell integrity and the mucosal barrier. Thus, HGF is widely available in the reproductive tract with functions that remain to be fully elucidated.

Human milk contains a complex uncharacterized, immune system able to exert actions both locally and systemically. This study reports the results of an ELISA-based quantitation of soluble Fas and soluble Fas ligand in human milk which may modulate the Fas/FasL system that is critical for the expression of immune tolerance and apoptosis. Production of Fas/FasL mRNAs by milk cells was also examined using RT-PCR. Fas is ubiquitously expressed in various cells and when bound by its ligand FasL, present predominantly on activated T- and NK cells, Fas-expressing cells are killed. A large amount of soluble Fas (1746-4320 pg/ml) is detected in colostrum, transitional milk and the mature milk of mothers delivering prematurely or at full-term, whereas FasL is present only in the range 123-310 pg/ml. Milk cells are positive for Fas mRNA, but negative for FasL mRNA. An excess of soluble Fas in human milk may bind to FasL preventing apoptosis and preserving epithelial barriers, and may represent an additional new mechanism whereby human milk favours immune tolerance and normal gastrointestinal development.

Marijuana, a widely abused drug in the US, and its derivatives (cannabinoids) have been used in AIDS and cancer patients for treatment of intractable nausea and cachexia. Yet, objective investigations of the effect of cannabinoids on the human immune system are few. We investigated the effect of delta9 tetrahydrocannabinol (THC) and cannabidiol (CBD) on cytokine production in vitro by human leukemic T, B, eosinophilic and CD8+ NK cell lines as models. THC decreased constitutive production of IL-8, MIP-1alpha, MIP-1beta, and RANTES and phorbol ester stimulated production of TNF-alpha, GM-CSF and IFN-gamma by NK cells. It inhibited MIP-1beta in HTLV-1 positive B-cells but tripled IL-8, MIP-1alpha and MIP-1beta in B-cells and MIP-1beta in eosinophilic cells but doubled IL-8. Both cannabinoids strongly inhibited IL-10 production by HUT-78 T-cells. Results indicate that THC and nonpsychotropic CBD have complex lineage and derivative specific effects on cytokines consistent with previous animal studies. These effects while of potential benefits in some inflammatory/autoimmune diseases may worsen HIV infection, tumorigenesis and allergic inflammation in the lung.

Purpose of Investigation: The hybrid capture human papillomavirus (HPV) DNA assay is offered by the manufacturer to assist clinicians with patients with ASCUS pap smear results to assess the risk factor and to potentially direct follow-up of these patients. In our practice, a gynecologic oncology practice that has a referral based population with abnormal pap smears, our purpose was to evaluate the patients referred with all grades of abnormal cervical cytology., Methods: One hundred consecutive patients who were referred for evaluation of abnormal cervical cytology: atypical squamous cells of undetermined significance (ASCUS); low-grade squamous intraepithelial lesion (LGSIL); high-grade squamous intraepithelial lesion (HGSIL); or squamous cell carcinoma (SCC) were evaluated by repeat pap smear, hybrid capture HPV DNA analysis and colposcopy. Colposcopic findings were recorded, and if appropriate, cervical biopsies were performed. Hybrid capture results were correlated with histologic and cytologic findings. Using histopathologic diagnosis as the reference standard, the sensitivity and positive predictive value of pap smear and high risk HPV were calculated. The Kappa test was used to correlate colposcopic and histopathologic findings., Results: Repeat pap smears at the time of initial consultation demonstrated 25 patients with normal results, 39 with LGSIL, 30 with HGSIL, 1 SCC and 5 ASCUS. Seventy-eight patients underwent cervical biopsy. Colposcopic findings correlated significantly with histopathologic findings (p<0.0001). Forty-four percent of patients tested positive for HPV DNA: 40 patients with high risk HPV, three patients with low risk HPV, and one patient with both high risk and low risk HPV. Sixteen of 39 patients (41%) with LGSIL on pap smear tested positive for high risk HPV; 37% of patients in this group required cervical conization because cervical biopsies demonstrated moderate/severe dysplasia. The diagnosis of moderate/severe dysplasia significantly correlated with the presence of high risk HPV [OR 78.9 (8.31-389.30)]. There was no significant correlation between the HPV DNA signal strengths and the histologic grade of dysplasia. The sensitivity and the positive predictive value of pap smear alone in identifying moderate/severe dysplasia was 62% and 96%, respectively. The combination of HGSIL pap smears and high risk HPV increased the sensitivity but not the positive predictive value for the detection of moderate/severe dysplasia to 77.7% and 95%, respectively (P=NS)., Conclusions: Although in this setting, the use of hybrid capture DNA testing did not significantly improve the sensitivity or positive predictive value of the diagnosis of HGSIL cytology when compared to cytologically indicated plus colposcopically directed cervical biopsies in this population of women at high risk for the presence of disease, the combination of HGSIL pap smears and high-risk HPV did result in a clinically important increase in the diagnosis of moderate/severe dysplasia.

Problem: How is it possible that the female genital tract immunologically does not reject spermatooa not the preimplantation and nidating embryo?, Methods: Four fluids of the human reproductive tract, i.e., human oviductal fluid (hOF), follicular fluid (FF), amniotic fluid (AF), and seminal plasma (SP) were investigated by specific ELISA for 18 cytokines. The concentrations, presence or absence of these compounds were evaluated for their possible role in the immunology of the reproductive process., Results: Stem cell factor and IL-11 were detected in all reproductive tract fluids examined whereas large amounts of IL-1 beta and IL-1RA was found in AF and hOF. Follicular fluid revealed IL-2. HOF contained IL 2, IL-6, IL-8, TNF-alpha, MIP-1 alpha, IFN-gamma, and high levels of IL-1 beta, IL-10, IL-1RA, and sIL-2R. Amniotic fluid contained sIL-2R, IL-8, IL-1 beta, IL-1RA, IL-6, TNF-alpha, and MIP-1 alpha. No IL-12 or IL-13 was detected in hOF follicular fluid or amniotic fluid. Almost no free TGF-beta 1 or TGF-beta 2 was found in any reproductive tract fluid except seminal plasma. Seminal plasma contained large quantities of free TGF-beta 1 (9,220 +/- 3,635 pg/mL) in addition to large quantities of latent TGF-beta 2 (2,933 +/- 2,169 pg/mL) and TGF-beta 1 (71,000 +/- 3,240 pg/mL). Furthermore, considerable concentrations of IL-8 (1900 +/- 374 pg/mL) and sIL-2R (350 mu/mL) exist in seminal plasma., Conclusions: HOF contains a high level of IL-10 (588 +/- 304 pg/mL), a powerful immune suppressor which probably plays a role in regulating immune responses in the fallopian tube and possibly in the endometrial cavity. Our observations suggest that seminal plasma with its huge content of TGF beta provides immune protection for sperm. Unfortunately, such high concentrations of TGF beta may also inhibit an immune defense in any organ in which semen is deposited.

To date no hematopoietic progenitors of dendritic Langerhans' cells (DLC), which represent an highly efficient class of antigen presenting cells, have been identified or the cytokines they elaborate have been defined. Here we describe an acute leukemia patient whose blasts (90-96% in peripheral blood and bone marrow) had a phenotype consistent with putative progenitors of DLC. The patient was treated with ara-C and VP-16 but did not achieve remission. The blasts had lobulated nuclei, no cytoplasmic vacuolation or Auer rods and were weakly positive for acid phosphatase and non-specific esterase and negative for PAS, granzyme A, dipeptidyl aminopeptidase IV, ATPase/ADPase and lysozyme production. The blasts were positive for CD1a, CD4, CD16, CD35, HLADR, HLADQ, CD11b, CD11c, CD14, CD33, CD34, CD11a, CD71, CD19, CD25, IL-2R beta and negative for CD2, CD7, CD8, CD10, CD22, CD56, CD57, surface or cytoplasmic CD3, TCR delta and TCR beta, HTLV-1p19 and P-glycoprotein. On liquid culture with or without 5 x 10(-9) M 12-O-tetradecanoylphorbol-13-acetate (TPA) for 3 days, the blasts formed aggregates of proliferating and elongating cells on the wall of the flasks with a decline in CD34, numerous dendritic processes appeared on the cells and there was strong positivity for ATPase/ADPase, but no other changes in phenotype. No macrophages were observed, indicating derivation from separate DLCs. Cytogenetic analysis showed chromosomal abnormalities and electron microscopy showed Birbeck granules. Southern blotting of DNA showed rearrangement of one allele for both JH and TCR beta but no HTLV-1 related sequences. Culture supernatants from blasts cultured with or without TPA showed the production of large amounts of IL-8, IL-6, TNF-alpha, MIP-1 alpha, IL-10 and interferon gamma and modest amounts of IL-1 alpha, GM-CSF and stem cell factor. The presence not only of CD1a, HLADR, HLADQ and many other characteristics including Birbeck granules, but also differentiation along the lines of DLC with appearance of dendritic processes on the cells and expression of ATPase/ADPase activity, indicate that the leukemic blasts in our patient represented a leukemic counterpart of normal progenitors of DLC and the leukemia a new entity which could possibly be classified as AML-M8. Lastly, many pro-inflammatory cytokines produced by DLC could contribute to inflammation and IL-10 to immunosuppression.

The culture supernatants from 43 human cell lines obtained during log phase and from purified normal peripheral blood B-lymphocytes cultured at 10(6) cells ml-1 for 48 h in RPMI 1640-5% fetal calf serum were examined for interleukin-8 (IL-8) using Elisa kits. Constitutive IL-8 production was found for 14/15 B-cell lines (5 derived from normal persons and 2 from AML patients, 1 pre-B-ALL, 2 CLL with trisomy 12, 2 HTLV-I+, 1 HTLV-II+, 1/2 Burkitt lymphoma), 4/16 T-cell lines (3/6 HTLV-I+, 1 HTLV-II+, 0/9 T-ALL), myeloid line HL-60, monocytoid line U937, 3/3 ovarian carcinoma, 1/1 endometriosis, 2/2 normal fibroblast, 0/2 C-ALL, 0/1 pre-erythroid line K562, as well as for normal B-lymphocytes. Later, cells examined by indirect immunofluorescence using IL-8 antibodies gave a positive reaction. DNA from 4 IL-8 producing and 3 non-producing cell lines, when probed with IL-8 cDNA gave the same 3.5 kb EcoRI fragment indicating similarities of the IL-8 gene in these cells. Two B-cell lines examined showed the expression of 1.8 kb IL-8 mRNA. These results indicate IL-8 production by a greater variety of cells than previously believed which open possibilities for new IL-8-mediated immune functions by such cells as B-cells.

Antibodies reactive with human T-cell leukemia virus type I (HTLV-I) proteins p19, p24, gp46, p56, and gp68 were detected in four of 27 patients with mycosis fungoides/Sézary syndrome (MF/SS) and one patient with Kaposi's sarcoma using radioimmunoprecipitation and Western blot analysis. Seroreactivity patterns to HTLV-I proteins of MF/SS sera were indeterminate or limited in comparison with sera of patients with adult T-cell leukemia/lymphoma. HTLV-I gag- and tax/rex-specific DNA was demonstrated in peripheral blood from three of the MF/SS patients and from the patient with Kaposi's sarcoma by the polymerase chain reaction. HTLV-I-specific DNA sequences were not detected in a cohort of seven seronegative MF/SS patients. The frequency of HTLV-I infection was four of 27 or 14.8% among the MF/SS patients, which is several hundredfold higher than in normal blood donors. The present data suggest a possible association of HTLV-I or a related retrovirus with mycosis fungoides/Sézary syndrome and Kaposi's sarcoma.

We used enzyme-linked immunosorbant assay (ELISA) and Western blotting, with "purified" human T-cell leukemia virus I (HTLV-I), to test for HTLV-I antibodies in 2583 plasma samples from 1053 leukemia/lymphoma patients treated at Roswell Park Memorial Institute, mostly between 1972 and 1984, and in 110 sera samples from normal healthy persons. The results demonstrate that ELISA and Western blot assay have limitations for HTLV-I antibody detection in an adult T-cell leukemia/lymphoma (ATL) nonendemic population. This conclusion is based on the many false reactives obtained by ELISA, and weak and indeterminate reaction (mostly p19 band) on Western blotting. All moderate to strongly HTLV-I ELISA-positive samples tested were negative for human immunodeficiency virus (HIV) antibodies. Although 6/27 mycosis fungoides (MF) patients tested gave mostly a weak reaction on HTLV-I ELISA, 3/6 MF patients gave multiple bands (p19, p31, p36, gp46) on Western blotting and three samples from one patient gave the same p31, p36, and gp46 bands. This may suggest involvement of some HTLV-I-related virus in MF. These results also indicate that prevalence of HTLV-I infection in leukemia/lymphoma patients was rare, if it exists at all, since, despite the reactivity of some sera with HTLV-I-suspected antigens, none of the samples satisfy the USPHS criteria for positivity which is based on the detection of antibodies to gag protein p24 and to an env gene product gp46 or gp61/68.

Normal and leukemic hematopoietic cell lysates were labeled with [3H]-diisopropylfluorosorophosphate ([3H]-DFP), an active site inhibitor of serine hydrolases. The labeled proteins in the lysates were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by counting of gel segments for radioactivity. The results indicate the presence of distinct [3H]-DFP binding patterns for different normal and leukemic hematopoietic cells; significantly lower labeling in normal or leukemic lymphoid cells compared to myeloid or monocytoid cells; lower labeling in acute myeloblastic leukemia (FAB-M1) as compared to acute myelomonocytic leukemia (FAB-M4), chronic myelomonocytic leukemia or monocytes and an increase in [3H]-DFP binding with cell maturation along granulocytic series. Thus, these patterns could be useful in discriminating acute lymphoblastic leukemia from myeloid/monocytoid types of leukemia and for following maturation of myeloid cells, and perhaps for studying functional or maturation defects in hematopoietic cells in other pathological conditions.

By means of the multiple marker analysis, a total of 55 human leukemia-lymphoma cell lines which included 15 T-cell, 30 B-cell, four myelomonocytic-cell, and six non-T, non-B cell lines was characterized for their marker profiles. The multiple markers used included a number of cell surface markers as detected by either rosette or immunofluorescence tests, enzyme assays, cytogenetic analysis, and certain functional assay. Based on the criteria previously defined it was found that all the cell lines were proved to represent original leukemia-lymphoma of ALL, AML, CLL, CML in blastic phase or variety of lymphomas. The monoclonality, a "frozen" state at a specific state of differentiation-maturation, and cytogenetic marker in each leukemia-lymphoma cell line were remarkable common properties and were stable for years of cultivation. Similar, if not identical, general characteristics were observed in the study on 344 cases of uncultured fresh leukemia-lymphomas by the multiple marker analysis. While no single marker specific to any type of tumor was found, the study offers not only a basis for better understanding of the biology of leukemia-lymphoma but also an insight into normal hematopoietic cell differentiation in man.

Fourteen streptovaricin derivatives were tested for inhibition of cellular nucleotide polymerases (deoxyribonucleic acid polymerases alpha, beta, and gamma, terminal deoxynucleotidyltransferase [TdT], and ribonucleic acid polymerase II), simian sarcoma virus deoxyribonucleic acid polymerase, and herpes simplex virus type 1-induced deoxyribonucleic acid polymerase (HSV-DP). Three compounds (strep-tovadienal C, prestreptovarone, and streptoval Fc) preferentially inhibited TdT and HSV-DP over the other enzymes. These compounds inhibited HSV-DP more potently than they inhibited TdT. Evidence indicated that the mode of inhibition of TdT and HSV-DP by streptovadienal C and prestreptovarone was by interaction with the enzymes and not with template-primer, initiator, substrates, or divalent cations required for enzyme activity. Furthermore, data suggested that these compounds bind with greater affinity to HSV-DP than to TdT. Streptovadienal C and prestreptovarone were examined for their effect on the replication of herpes simplex virus type 1 in African green monkey kidney (CV1) cells. These compounds produced 2- and 3-log drops in virus titer, respectively, at concentrations not significantly affecting cell viability. This correlated with evidence indicating a greater binding affinity of these compounds for HSV-DP over cellular nucleotide polymerases.

Although leukocytes from all 13 acute lymphoblastic leukemia patients examined had high terminal deoxynucleotidyl transferase (terminal transferase) activity (20 to 100 units/mg of cellular DNA, where 1 unit equals 1 nmole of nucleotide polymerized in 1 hr) and those from 21 acute myelocytic leukemia patients had low terminal transferase activity (0.2 to 2 units/mg of cellular DNA), the bone marrow and peripheral blood leukocytes from 2 patients with acute myelocytic leukemia, diagnosed on the basis of clinical features and the morphology, cytochemistry, and cytogenetics of the leukemic cells, had terminal transferase activity (39 to 52 units/mg of cellular DNA) equivalent to that found in leukemic lymphoblasts. These results bring under question the specificity of high terminal transferase activity outside of the thymus as a marker for leukemic lymphoblasts and, secondarily, the derivation of acute lymphoblastic leukemia cells in all cases from thymocytes. Perhaps malignant transformation in a pleuripotent stem cell with derepression of the genome for terminal transferase could account for high terminal transferase activity observed in certain leukemic cells.

Antibodies reactive against human T-cell leukemia virus I (HTLV-I) were detected by indirect immunofluorescence assay using MT-2 as target cells, enzyme linked immunosorbent assay screen and competition assay, and Western blot analysis in three sera (one collected in 1979) from a captive gorilla which developed diffuse histiocytic lymphoma in 1983. The sera from four other healthy gorillas housed separately were HTLV-I antibody negative. All sera were negative for HTLV-III antibodies by enzyme linked immunosorbent assay. Southern blot analysis of DNA from lymphoma tissue after digestion with BamHI and using complete HTLV-I genome probe gave one 10-kilobase fragment and a characteristic 1.05-kilobase internal fragment detected in all known HTLV-I isolates. These results indicate that the gorilla was infected with HTLV-I or a closely related simian virus several years before the development of lymphoma.

Terminal deoxynucleotidyl transferase activity is as much as several hundred fold greater in acute leukemic lymphoblasts than in unstimulated or phytohemagglutinin stimulated normal lymphocytes, and in leukocytes from other leukemias or from acute lymphoblastic leukemia patients in remission. On the other hand, acute leukemic lymphoblasts and myeloblasts as well as phytohemagglutinin stimulated normal lymphocytes have DNA polymerase activity an order of magnitude higher than that in unstimulated normal lymphocytes, and in leukocytes from chronic leukemias or from acute lymphoblastic leukemia patients in remission. Terminal deoxynucleotidyl transferase thus appears to be a useful index for the detection of leukemic lymphoblasts.

DNA or chromatin from young (7 day) first seedling leaves of barley showed only one component which migrated little into 1% agarose gels on electrophoresis. However, DNA or chromatin from senescent (17-, 19-, and 23-day-old) leaves showed additional dispersed components migrating throughout the length of the gel which increased with age. These low molecular weight components increased even more on autodigestion of chromatin from senescent leaves by its associated DNase or by digestion of DNA from senescent leaves with partially purified chromatin DNase. DNA or chromatin from young leaves also produced gel pattern similar to old leaves on digestion with partially purified chromatin DNase from old barley leaves. Thus, fragmentation of DNA and chromatin by chromatin associated DNase, previously shown to increase 4000% on aging, occurs during senescence in barley leaves.

Two unique cell lines, NALM-1 and BALM-2 derived from lymphoblast-like cells of chronic myelogenous leukemia and rare B cell acute lymphoblastic leukemia patients, respectively, were compared with fresh parent cells from the patients and with a Philadelphia chromosome positive K-562 cell line previously established from a chronic myelogenous leukemia patient in blastic phase. NALM-1 resembled the parent cells in the presence of Philadelphia chromosome, non-T/non-B acute lymphoblastic leukemia specific antigens and lack of T or B cell markers, whereas BALB-2, like the parent cells, had two chromosome markers and bore kappa, delta and mu immunoglobulins. NALM-1 lacked Epstein-Barr virus genome, whereas BALM-2 showed the presence of Epstein-Barr virus genome. K-562 cells lacked all the antigen markers examined. All cells had high DNA polymerase alpha activity and low DNA polymerase gamma activity. NALM-1, like the parent cells and unlike K-562 cells, had high terminal deoxynucleotidyl transferase activity of about 200 mu/mg DNA, whereas BALM-2, like its parent cells, had terminal deoxynucleotidyl transferase activity of 1-2 mu/mg DNA (1 u = 1 nmole Mn++-dGTP/h on dA12-18 initiator). Terminal deoxynucleotidyl transferase was characterized by its chromatographic and sedimentation behavior, thermal sensitivity and specific inhibition by streptolydigin and terminal deoxynucleotidyl transferase antisera. These results indicate that NALM-1 and K-562 may represent different phenotypes of cells in CML blastic crisis. Moreover, NALM-1 and BALM-2 seem to have retained the characteristics of original leukemic cells from which they may have been derived.

Forty-seven human leukaemia/lymphoma cell lines belonging to myelocytic, monocytic, non-T/non-B, T-, and B-lineage and representing different levels of maturation as well as fresh cells from normal and leukaemic subjects were examined for immunological markers and cytochemically for acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase (pH 5.8 and 8.0), alpha-naphthyl butyrate esterase (pH 5.8 and 8.0), non-specific esterase, chloroacetate esterase, chymotrypsin-like protease, deoxyribonuclease II, beta-glucuronidase, sudan black, and periodic acid Schiff's staining. Strong sudan black, nonspecific esterase, and chloroacetate esterase reaction was obtained only for myelocytic and monocytic cell lines with the reaction intensity increasing progressively in more mature cells. Focal acid phosphatase reaction like T-ALL was found in all T-ALL cell lines, whereas myeloid/monocytoid lines had semicircular distribution and B-cell lines cytoplasmic distribution of activity. Acid phosphatase activity appeared to decline with maturation along both myeloid and T-cell lineage. High activity of alpha-naphthyl acetate esterase and alpha-naphthyl butyrate esterase both at pH 5.8 and 8.0 and of beta-glucuronidase was found in myeloid/monocytoid lines although both B- and T-cell lines in contrast to peripheral blood B-cells also had significant esterase activity. alpha-Naphthyl butyrate esterase activity declined with increasing cell maturation along myeloid lineage. Except for weak activity in two B-cell lines alkaline phosphatase was not detected in any cell lines. Monocyte esterase activity was inhibited by sodium fluoride whereas acid phosphatase, only from hairy cell leukaemia line, was resistant to L-tartarate. Although periodic acid Schiff's staining could not distinguish myeloid, T-, B-, or non-T/non-B cell lines it gave characteristic reaction (large number of coarse granules against a clear background forming a ring around the nucleus) with erythroblastic leukaemia cell line and along myeloid series its intensity increased in more mature cells. Deoxyribonuclease II and chymotrypsin-like protease staining were not discriminatory. The results of this study show that cytochemical staining characteristics of various leukaemia/lymphoma cell lines are comparable to those of corresponding cells from patients and that the intensity and pattern of expression of these activities are related to cell type and degree of cell maturation. These studies give further credence to the use of these cell lines in cell differentiation, differential drug cytotoxicity, and many other studies.

All 5 thymus-dependent cell (T-cell) lines (Molt-3; Molt-4; RPMI-8402; CCRF-CEM; CCRF-HSB-2) and 7 thymus-independent cell (B-cell) lines (RPMI-8382, RPMI-8392, RPMI-8412, RPMI-8422, RPMI-8432, RPMI-8442, CCRF-SB) established so far from acute lymphoblastic leukemia patients were examined for deoxynucleotide polymerizing enzymes. All T- and B-cells had DNA polymerase gamma, DNA polymerase beta, and terminal deoxynucleotidyl transferase both in the soluble (the latter 2 enzymes only in small amounts) and chromatin fraction, whereas DNA polymerase alpha was found only in the soluble fraction. With respect to their sedimentation and chromatographic behavior, template-primer requirements, Km for deoxythymidine triphosphate or deoxyguanosine triphosphate divalent cation preference, effect of NaCI and inhibitors, the enzymes from T- and B-cells resembled each other and those from other mammalian cells. DNA polymerase alpha, beta, and gamma from T-cells like those from "fresh" acute lymphoblastic leukemia cells, were more thermolabile than those from B-cells or phytohemagglutinin-stimulated normal lymphocytes. In addition, the terminal deoxynucleotidyl transferase from the above cells was completely inactivated in 5 to 6 min at 50 degrees, whereas the DNA polymerase alpha, beta, and gamma retained considerable activity even after heating for 25 min at 50 degrees. DNA polymerase activity of the soluble fraction from T-cells was of the same magnitude as in B-cells when expressed on a DNA basis but twice that of B-cells when expressed on a protein basis. High terminal deoxynucleotidyl transferase activity, equivalent to that observed in acute lymphoblastic leukemia cells, was found in all T-cell lines that, when expressed on a DNA basis, was 30 to 100 times higher than the B-cell lines tested. These results support the suggestion of earlier investigators that T-cell lines examined here may have originated from leukemic cells.

We have compared the relative inhibitory activity of poly (A) with its analogues poly N6-isopentenyl adenylic acid (poly(i6 A)) and poly N6-benzyl adenylic acid (poly(bzl6A)), and of poly (U) with its analogue poly 2'-fluoro-2'-deoxyuridylic acid (poly(dUfl)), against DNA polymerase, alpha, beta and gamma and terminal deoxynucleotidyl transferase from human cells and two oncorna virus DNA polymerases. Although poly (A) and its analogues were equally inhibitory against endogenous RNA-directed DNA polymerases of murine and feline leukemia viruses, the analogues in contrast to poly (A) were strongly inhibitory against all four cellular enzymes. Poly (dUfl), on the other hand, was up to 100-fold more potent than poly (U) against both viral and cellular enzymes. Since poly (U) at 100 mug/ml and poly (dUfl) at 1 mug/ml had no effect on terminal deoxynucleotidyl transferase while inhibiting other enzymes by 80--100 per cent these polymers could be useful in the characterization and assay of terminal deoxynucleotidyl transferase. In addition, the polymers such as poly (igA) and poly (bzl5A) which were strongly inhibitory to all cellular enzymes, could be useful in cancer chemotherapy if taken up preferentially by the malignant calls due to their high pinocytic activity. The results also demonstrate potential for large variation in inhibitory activity of polyribonucleotides as related to their chemical composition.

The cell membrane fraction from c-ALL, B-ALL, Ph' + ALL, B-CLL, T-CLL, AML, blastic-CML, normal leukocytes, PHA-stimulated lymphocytes and several T, B and myeloid human leukemic cell lines has been used in different cell types to demonstrate different patterns of glycosyltransferase activity. Both B- and T-CLL cell membranes have low fucosyltransferase B and A activity compared to acute leukemias; while sialyltransferase activity is higher in B- than in T-CLL. AML cell membranes and ML-1 human myeloblast cell line membranes have exceptionally high fucosyltransferase A activity compared to all other leukemic cells or cell lines. Human leukemic B cell lines expressed cell membrane sialyltransferase, fucosyltransferase B and probably fucosyltransferase A activity several times higher than T cell lines. Human myeloid cell lines ML-1 and HL-60 express 5- to 20-fold higher galactosyltransferase activity than human leukemic T and B cell lines. Both sialyltransferase and galactosyltransferase activity were higher in all leukemic cells than in normal leukocytes and PHA-stimulated normal lymphocytes. This is the first study carried out on glycosyltransferases using cells obtained from leukemic patients characterized immunologically. These results indicate that all glycosyltransferase activity, with the exception of fucosyltransferase activity in CLL, were higher in leukemic cells than in normal cells. Moreover, large differences in these enzymes, e.g. very high galactosyltransferase activity in myeloid cell lines compared to B and T cell lines, of fucosyltransferase A in AML and myeloblast cell lines compared to all other cells, and of sialyltransferase in B-CLL or B cell lines compared to T-CLL or T cell lines, could be useful in characterizing certain leukemias and hematopoietic cell lines.

Effect of dexamethasone of the growth of two T-cell acute lymphoblastic leukemia (T-ALL) cell lines, JM and TALL-1 and an American Burkitt lymphoma cell line, Ramos, of B-cell type was examined. While the growth of both TALL-1 and Ramos cell lines was significantly inhibited by dexamethasone at the concentration of 10(-7) M, JM cell line was markedly resistant at the high concentration of 10(-5) M of dexamethasone. The assay of glucocorticoid receptors showed that a small amount of the binding site was found in both T-cell leukemia cell lines, JM and TALL-1 and that a moderate amount of the binding site was found in Ramos cell line. Thus, glucocorticoid sensitivity (growth inhibition of these leukemia-lymphoma cell lines by dexamethasone) did not correlate with either the number of glucocorticoid receptor or with surface membrane markers of the target cells. This result suggests further marked heterogeneity of leukemic cells.

Permanent human hematopoietic cell lines representing T-cell, B-cell and non T/non B (null-cell) leukemia have been established. Comparative analyses were made for their phenotype characteristics. A number of characteristics common within the 7 T-cell lines studied or distinct from other leukemia-type lines were described. Usefulness, validity and limitation of these findings are discussed in connection to the attempt at classification of ALL, CLL and blastic phase of CML. The great majority of CLL were SmIg+-B-cell leukemia and a single case of T-cell CLL was documented. Except 10% as T-cell ALL and a single case of B-cell ALL, the majority of ALL were found to be the non T/non B ALL. Nevertheless, little evidence was suggested from the present study in favor for a notion that the T-cell ALL and the non T/non B ALL are two distinct diseases.

Pyrans are co-polymers of divinyl ether and maleic anhydride. Four pyrans of various molecular weights more potently inhibited terminal deoxyribonucleotidyltransferase (EC 2.7.7.31) from a human cell line of acute lymphoblastic leukemia origin (Molt-4) than they did DNA polymerases alpha, beta and gamma from these cells and DNA polymerase from simian sarcoma virus. For example, the concentrations of one pyran required for 50% inhibition of terminal deoxynucleotidyltransferase, DNA polymerases alpha, beta and gamma and viral DNA polymerase were 0.9, 110, 125, 35 and 47 microgram/ml respectively. Quantitatively similar results were obtained with the other pyrans. Inhibition of these enzymes by pyran was dependent on the concentrations of both the bivalent cation and template/primer or initiator in assay mixtures, but not on the concentrations of the substrate (deoxyribonucleoside 5'-triphosphate), enzyme, or bovine serum albumin. These results suggested that pyran inhibited these enzymes by complexing bivalent cations, which caused a decreased affinity of template/primer or initiator for each enzyme and a decrease in enzyme activity.

We report a case of acute leukaemia with the t(4;11) chromosomal translocation which, at initial diagnosis, had L-1 lymphoblasts that were positive for terminal deoxynucleotidyl transferase (TdT) and HLA-DR but negative for myeloid cytochemical markers. At last relapse the patient had mostly monocytoid blasts which were not TdT negative but were positive for HLA-DR, weakly positive for Sudan Black B (SB), periodic acid Schiff's (PAS), naphthol AS-D acetate esterase (NSE), chloroacetate esterase (CAE) and negative for acid phosphatase (AP) and nitroblue tetrazolium (NBT) reduction. Treatment with 12-o-tetradecanoylphorbol-13-acetate (TPA) in vitro induced differentiation to macrophage-like cells that were strongly positive for SB, PAS, NSE, AP, CAE and NBT reduction, indicating a latent monocyte-like phenotype. Thus the leukaemic cell clone or a precursor clone with the t(4;11) translocation manifested a lymphoid phenotype at initial diagnosis and a monocytoid phenotype at relapse. Immunoglobulin gene analysis of the monocytoid relapse blasts revealed rearrangements of the heavy chain gene alleles and germline light chain genes. Thus, the leukaemia clone with the t(4;11) chromosomal translocation could be a bipotential cell with heavy chain gene rearrangements occurring in a primitive cell which may retain the ability to differentiate along the myeloid-monocytoid lineage in response to the appropriate stimulus. Alternatively, these characteristics may result from a transformation associated event.

Specificity of TdT5 as a marker for ALL was evaluated by determining its activity in cells from normal control subjects and from 35 pediatric patients with ALL, AML, Hodgkin's disease and disseminated Burkitt's lymphoma. We evaluated the DNA polymerase activity, cell surface phenotypes (E rosettes, EAC rosettes, Smlg and la-like, HTLA and cALL antigens), and hematological and cytochemical characteristics in both the normal and patient groups. DNA polymerase alpha + beta and DNA polymerase gamma activity were indiscriminately high in all immature cells as found in ALL, AML, Burkitt's lymphoma and phytohemagglutinin-stimulated normal lymphocytes, when compared to mature leukocytes found in normal individuals or in patients whose cancer was in remission. High TdT activity was found in 24 of 26 T and non-T/non-B ALL patients in active phase as well as in two of three AML patients one of whom had Auer rods. Thus, TdT, although valuable for monitoring ALL patients, may have limitations in separating AML from ALL.

DNA polymerases alpha and beta from Molt-4 cells are inhibited by bleomycin, whereas DNA polymerase gamma assayed with poly-(A)-(dT)12-18 as the template primer or terminal deoxynucleotidyl transferase assayed with activated DNA, poly(dA), (dG)12-18 or (dA)12-18 as the initiator are not inhibited by this antibiotic. Inhibition by bleomycin increased the Km for template DNA but not that for dTTP. Increasing amounts of bleomycin did not affect the Vmax for DNA polymerase alpha or beta when the amount of template DNA was varied but it reduced the Vmax for these enzymes when dTTP was varied. Moreover, the addition of extra template reversed the bleomycin inhibition but the addition of extra enzyme did not. Although dithiothreitol was required for bleomycin inhibition of DNA polymerase activity, bleomycin preincubated with dithiothreitol (or beta-mercaptoethanol) at pH 6.5 to 9.0 lost its inhibitory activity. This was not the case when DNA was also included in the preincubation mixture. The results obtained in this study indicate that bleomycin inhibits DNA polymerases alpha and beta by a thiol reagent-dependent interaction with the template. Thus, the antitumor activity of bleomycin may be greatly influenced by the concentration of sulfhydryl compounds and their proximity to DNA in the target cells.

Although the relative incorporation of incorrect nucleotide (dCTP or dGTP) into poly(dA-dT).poly(dA-dT) by partially purified 3-4S DNA polymerase from normal or leukaemic human cells was four to five times higher than that by the 6-7S DNA polymerase, no significant differences in the infidelity of these polymerases between normal and leukaemic cells were noted.

Restriction endonuclease MspI digested significantly more than HpaII the DNA and chromatin from normal and leukemic human cells, although both enzymes digested DNA more than chromatin. Moreover, DNA and chromatin from normal cells showed higher digestion by HpaII compared to DNA and chromatin from leukemic cells indicating higher frequency of Cm5CCG in latter DNA. EcoRII and BstNI, which have the recognition sequence CCATGG but cut at different points, digested all DNAs significantly, as did BstNI for chromatin from all sources. However, chromatin from normal cells showed only limited digestion by EcoRII which significantly digested chromatin from leukemic cells. This could result from subtle differences in the conformation of normal and leukemic cell chromatin involving recognition sequences for EcoRII.