PCR based diagnostic assay targeting the beta tubulin gene for the detection of Trichomonas vaginalis infection in vaginal swab samples of symptomatic and asymptomatic women in India

ObjectiveTo develop an in-house PCR based diagnostic assay for identification of strains isolated from symptomatic and asymptomatic subjects of India, targeting the β-tubulin gene using specific primers.MethodsIn the present study a primer set is designed to target a well-conserved region in the beta-tubulin gene of Trichomonas vaginalis (T. vaginalis). All strains of T. vaginalis were tested and successfully detected by PCR yielding a single predicted product of 198 bp in gel electrophoresis, while there was negative response with DNA from Giardia lamblia, Toxoplasma gondii, Leishmania donovani and Entamoeba histolytica. The sensitivity and specificity for a single T. vaginalis cell per PCR was achieved. Axenic Culture, performed with long term axenized T. vaginalis culture system, was routinely examined to identify T. vaginalis.ResultsThe PCR based investigations with 498 vaginal swab samples from women attending OPD clinics of Halberg Hospital Moradabad and Queen Mary's Hospital, Lucknow, India and 17 long term axenic cultures maintained at PGIMER, Chandigarh, India using primer set BTUB 1 & BTUB 2 showed sensitivity and specificity response of 98% and 100%, respectively, while wet preparation in clinically isolated samples responded up to 62.5%. The PCR product sequencing result of symptomatic strains (SS1) of T. vaginalis (744 bp long) was submitted to NCBI (Accession No: JF513200). It shows maximum identity 98 % with XM_001284521 Trichomonas vaginalis G-3 beta-tubulin (btub) putative partial mRNA.ConclusionsThe data gathered in the present study entail that the diagnosis of T. vaginalis infection by PCR may be established as a sensitive and specific protocol, to be incorporated into a joint strategy for the screening of multiple STDs by employing molecular amplification technique. The merits and precautions of the protocol have been discussed.     

18S ribosomal DNA based PCR diagnostic assay for Trichomonas vaginalis infection in symptomatic and asymptomatic women in India

ObjectiveTo identify the cases of trichomoniasis in symptomatic and asymptomatic Trichomonas vaginalis (T. vaginalis) infected patients by PCR amplification of hypervariable 18S rRNA gene and to assess the sensitivity of restriction fragment length polymorphism (RFLP) technique for their diagnosis.MethodsWe enrolled 498 women of child bearing age groups, with their pre-informed consent, attending OPD for their routine checkups and STI related problems. Trichomoniasis was diagnosed on the basis of wet mount preparations and PCR with a primer set targeting a well-conserved region in the 18S rRNA genes of T. vaginalis, respectively. Sequencing was done for differentiating the symptomatic and asymptomatic strains of axenic and clinical isolates.ResultsAfter PCR diagnosis T. vaginalis infection was detected in 17 (3.42%) out of 498 clinical isolates. Seventeen axenic and sixteen clinical strains of T. vaginalis tested were successfully detected by PCR yielding a single predicted product of 312 bp in gel electrophoresis followed by restriction digestion with restriction endonuclease HaeIII. After restriction digestion they gave two bands, one of 101 and the other of 211 bp, while there was negative response with DNA from Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Toxoplasma gondii, Leishmania donovani and Entamoeba histolytica. An optimal analytical sensitivity and specificity of one T. vaginalis organism per PCR was achieved. Sequence of symptomatic and asymptomatic strains of axenic and clinical isolates are somewhat differentiated on the basis of point mutations in their 18S rRNA gene.ConclusionsOnly few factors are known to predict symptoms of T. vaginalis infection, although the majority of women are infected with trichomoniasis are reported. Therefore the application of sensitive PCR based diagnosis may be quite useful for routine diagnosis of T. vaginalis strains.     

Biliary strictures after liver transplantation: Clinical picture, correlates and outcomes

We retrospectively examined 154 adults to ascertain the frequency, site of and pre-disposing factors for biliary strictures after liver transplantation, as well as their management and clinical outcome. Twenty patients (12.5%) were identified with biliary strictures; 16 were non-anastomotic and four were anastomotic strictures. The median time from transplantation to stricture diagnosis was 17 weeks (range 3–366). Of the 16 non-anastomotic strictures, six were intrahepatic, eight hilar and two extrahepatic (donor bile duct). A control group (n = 32) of patients transplanted immediately before and after index cases was used to examine for correlates in patients with non-anastomotic strictures. At the time of diagnosis in the non-anastomotic index cases, there was a higher incidence of: (i) biliary sludge (63 vs 0%; P< 0.001); and (ii) clinical cholangitis (75 vs 0%; P< 0.001) compared with controls. Primary sclerosing cholangitis was more often the diagnosis in index patients with non-anastomotic strictures compared with controls (31 vs 9%; P<0.05). There were no differences between index patients and controls (non-anastomotic group) in ABO blood group non-identity, cold allograft ischaemia time, use of OKT3 (murine monoclonal antibody to CD³) and hepatic artery thrombosis. Of 15 patients treated with balloon dilatation, seven required stent insertion although none have required surgery. As determined by liver function tests, there was evidence of persisting graft dysfunction in index patients compared with controls (SAP 381 vs 112 U/L, P< 0.001; GGT 529 vs 80 U/L, P< 0.001), but there was no difference in survival during a median follow-up time of 16 months (range: 3–48 months) from stricture diagnosis. In conclusion, biliary strictures tend to occur within 6 months of transplantation and are an important cause of ongoing graft dysfunction. Non-anastomotic strictures were more common in patients requiring transplantation for primary sclerosing cholangitis.     

Dose-response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates

Thrombopoietin (TPO) is the physiologic Mpl-ligand regulating platelet production. Pegylated human recombinant megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated polypeptide Mpl-ligand derivitized with poly-(ethylene glycol), induces megakaryocyte endoreduplication and proliferation in vitro and in vivo. In the present study, the dose-response effects of PEG-rHuMGDF on pharmacokinetics, megakaryocytopoiesis, platelet production, and platelet function were characterized for dosing 0.05, 0.10, 0.50, or 2.5 micrograms/kg/d in 22 baboons for 28 days. Daily subcutaneous injections of PEG-rHuMGDF produced linear log-dose responses in (1) steady-state trough plasma levels of PEG-HuMGDF (P < 10(-3)); (2) marrow megakaryocyte volume (P < 10(-3)), ploidy (P < 10(-4)), and number (P < .01); and (3) peripheral platelet concentrations (P < 10(- 4)) and platelet mass turnover (P < 10(-3)). Platelet morphology, life span, and recovery were normal, and peripheral leukocyte, neutrophil, and erythrocyte counts were not significantly affected by PEG-rHuMGDF (P > .1 in all cases). PEG-rHuMGDF at 0.5 micrograms/kg/d produced similar blood concentrations of Mpl-ligand and platelets as 10 times the dose of rHu-MGDF (5.0 micrograms/kg/d), reflecting the extended plasma half-life achieved through pegylation. Whereas PEG-rHuMGDF did not induce platelet aggregation in vitro, platelet aggregatory responsiveness induced by thrombin receptor agonist peptide (TRAP1-6) and collagen was transiently enhanced ex vivo during the initial few days of PEG-rHuMGDF administration. However, adenosine diphosphate (ADP)-induced platelet aggregation was not enhanced ex vivo by PEG- rHuMGDF therapy. 111In-platelet deposition on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts increased in direct proportion to the circulating platelet concentration (P < 10(-4) for both EA and VG); 125l-fibrin accumulation was not affected by PEG-rHuMGDF-induced increases in peripheral platelet counts. Changes in platelet production and function produced by PEG-rHuMGDF returned to baseline within 2 weeks after discontinuing treatment. Thus, in nonhuman primates, PEG- rHuMGDF increases platelet production in a linear log-dose-dependent manner by stimulating megakaryocyte endoreduplication and new megakaryocyte formation from marrow hematopoietic progenitors. These findings suggest that appropriate dosing of PEG-rHuMGDF therapy during periods of chemotherapy-induced marrow suppression may maintain hemostatic concentrations of peripheral platelets without increasing the risk of thrombosis.     

Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu- MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5- fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating concentration of platelets for both EA and VG (r=.98 in both cases), without significant changes in fibrin accumulation (P > .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1-6) (P < .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P > .5), rHu-MGDF enhanced aggregatory responses to low doses of physiologic agonists, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P- selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.     

CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa

Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with greater than or equal to 95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1-1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1-1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1-1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.     

Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia

The chimeric BCR/ABL protein is characteristic of Philadelphia (Ph)+ leukemia because it is the direct product of the Ph translocation and it has been shown to play a causal role in the genesis of leukemia. The BCR/ABL protein exhibits a deregulated tyrosine-kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. CRKL, an adaptor protein consisting of SH2 and SH3 domains in the absence of a catalytic domain, is one potential in vivo substrate of BCR/ABL. Previous experiments have shown that CRKL is phosphorylated on tyrosine in the chronic myelogenous leukemia (CML) cell line K562 and that CRKL is a substrate for ABL and for BCR/ABL in COS-1 cells. In the current study, we show that in peripheral blood cells a direct correlation exists between the presence of BCR/ABL and the phosphorylation status of CRKL. In Ph- peripheral blood cells, CRKL is present only in the nonphosphorylated form. In contrast, all BCR/ABL+ CML and acute lymphoblastic leukemia patient samples examined showed clear tyrosine-phosphorylation of CRKL. This result strongly suggests that CRKL is a biologically significant substrate for BCR/ABL and is likely to play a major role in the development of Ph+ leukemia.     

BCR-ABL protein expression in peripheral blood cells of chronic myelogenous leukemia patients undergoing therapy

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome (Ph1) in more than 95% of these patients. The Ph1 and the resulting BCR-ABL fused genes are markers for this type of leukemia. In CML, the product of the fused BCR- ABL gene is typically a protein of approximately 2,000 amino acids termed P210 BCR-ABL. We have developed an assay for the BCR-ABL protein involving Western blotting of circulating white blood cells (WBC) with an anti-ABL monoclonal antibody that can detect P210 BCR-ABL and P145 ABL in peripheral blood cells from chronic phase Ph1-positive leukemia patients. This assay was used to analyze the BCR-ABL protein content of circulating WBC from CML patients before and after various treatments. In parallel to changes in percentages of Ph1-positive blood cells as determined by cytogenetic analyses of bone marrow samples, BCR-ABL protein expression in blood cells decreased or increased as patients entered remission or underwent relapse. Of interest, six Ph1-negative CML patients were BCR-ABL protein-positive. All except one had a rearrangement in the major breakpoint cluster region and that patient expressed P185 BCR-ABL and not P210. Our results indicate that the BCR- ABL Western blotting assay has clinical applications for both diagnosis and prospective evaluation of Ph1-positive and Ph1-negative CML patients.     

Evaluation of abdominal trauma by computed tomography

Computed tomography (CT) was used in the evaluation of 100 patients suffering abdominal trauma. The type of trauma was blunt in 78 patients, penetrating in eight, and iatrogenic in 14. Forty per cent of cases had normal CT scans, while 60% showed substantial abdominal or retroperitoneal injuries. Surgery, clinical follow-up, and repeated radiologic examinations confirmed the accuracy of CT, and there were no cases in which medical or surgical management was inappropriately guided by CT. A wide variety of injuries was detected, including 19 splenic, eight hepatic, six pancreatic, 13 renal, 13 retroperitoneal or abdominal wall, and one intraperitoneal. CT has major advantages over plain radiography, radionuclide imaging, and angiography in assessment of trauma-induced injuries.     

Characterization of a suppressor T-cell chronic lymphocytic leukemia with ADCC but not NK activity

A patient with T-cell chronic lymphocytic leukemia (T-CLL) is reported whose cells demonstrate in vitro suppression of normal lymphocyte mitogen stimulation. The patient, who remains in Rai's clinical stage 0 on no therapy after more than 24 mo of observation, has shown a less aggressive clinical course than is usually attributed to T-CLL. His peripheral blood lymphocytes (PBL) were characterized by functional assays as well as surface markers. Over 90% of the patient's PBL formed rosettes with sheep erythrocytes and were lysed by two T-cell-specific antisera plus complement, while less than 1% bore surface immunoglobulins, and only 3% had complement receptors. In addition, 45% of the PBL demonstrated Ia-like antigens, more than 50% expressed a receptor for the Fc portion of IgG(T gamma), and most of the sheep erythrocyte rosettes were inhibited by theophylline. The patient's cells failed to respond to several mitogens and they caused marked suppression of lymphoproliferative responses to normal PBL to phytohemagglutinin (PHA) and concanavalin A (Con-A). The patient's lymphocytes also exhibited antibody-dependent cytotoxic activity (ADCC) against antibody-coated nucleated target cells, but lacked demonstrable natural killer (NK) activity. This patient's T-CLL cells appear to represent the clonal expansion of a subset of T cells with a previously undescribed pattern of suppressor and cytotoxic activities.