Qualitative Reverse Transcriptase-polymerase Chain Reaction Research Articles

SARS-COV2 Virus in Tears and Conjunctival Secretions of COVID-19 Patients

Purpose: To determine the presence of SARS-COV 2 viruses in tears and conjunctival secretions of patients with diagnosed COVID-19.
 Study Design: Prospective interventional case series.
 Place and Duration of Study: Rawalpindi Institute of Urology, Department of infectious diseases, Holy Family Hospital, Rawalpindi Medical University, from September 2020 to October 2020.
 Methods: Samples of conjunctival and tear secretions were collected from 60 hospitalized patients of COVID-19 who were confirmed with nasopharyngeal swabs test. Disposable conjunctival swab sticks were used for sampling. Samples were taken within 3 days of admission. SARS-COV 2 virus evaluation in tears and conjunctival secretions was done by Qualitative RT-PCR (Reverse transcriptase polymerase chain reaction) analysis. Ocular features were documented. Regarding systemic course of illness, details were noted from their hospital records.
 Results: Mean age of the patients was 56.63 ± 16.373 years. Out of 60 patients, 42 were males and 18 were females. Twenty two (36.6%) patients had mild disease and moderate and severe disease was present in 19 patients (31.6%) each. Six (10%) patients had positive conjunctival and tear secretions for SARS-COV 2 viral RNA. All patients with positive ocular secretions for viral RNA were in first week of course of disease and 3 patients had severe COVID 19 disease signifying high viral load. Only one patient had conjunctivitis and ocular symptoms of redness and foreign body sensation.
 Conclusion: There is likelihood of SARS-COV 2 virus transmission via ocular secretions as frequency of patients with SARS-COV 2 viral RNA detected in tears was 10% in current study therapeutic services.
 Key Words: SARS-COV 2 Virus, COVID 19, Conjunctivitis, Qualitative RT-PCR, Ocular Manifestations

Abelson interactor 1 splice isoform-L plays an anti-oncogenic role in colorectal carcinoma through interactions with WAVE2 and full-length Abelson interactor 1.

BACKGROUNDExpression of the full-length isoform of Abelson interactor 1 (ABI1), ABI1-p65, is increased in colorectal carcinoma (CRC) and is thought to be involved in one or more steps leading to tumor progression or metastasis. The ABI1 splice isoform-L (ABI1-SiL) has conserved WAVE2-binding and SH3 domains, lacks the homeo-domain homologous region, and is missing the majority of PxxP- and Pro-rich domains found in full-length ABI1-p65. Thus, ABI1-SiL domain structure suggests that the protein may regulate CRC cell morphology, adhesion, migration, and metastasis via interactions with the WAVE2 complex pathway.AIMTo investigate the potential role and underlying mechanisms associated with ABI1-SiL-mediated regulation of CRC.METHODSABI1-SiL mRNA expression in CC tissue and cell lines was measured using both qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR. A stably ABI1-SiL overexpressing SW480 cell model was constructed using Lipofectamine 2000, and cells selected with G418. Image J software, CCK8, and transwell assays were used to investigate SW480 cell surface area, proliferation, migration, and invasion. Immunoprecipitation, Western blot, and co-localization assays were performed to explore intermolecular interactions between ABI1-SiL, WAVE2, and ABI1-p65 proteins.RESULTSABI1-SiL was expressed in normal colon tissue and was significantly decreased in CRC cell lines and tissues. Overexpression of ABI1-SiL in SW480 cells significantly increased the cell surface area and inhibited the adhesive and migration properties of the cells, but did not alter their invasive capacity. Similar to ABI1-p65, ABI1-SiL still binds WAVE2, and the ABI1-p65 isoform in SW480 cells. Furthermore, co-localization assays confirmed these intermolecular interactions.CONCLUSIONThese results support a model in which ABI1-SiL plays an anti-oncogenic role by competitively binding to WAVE2 and directly interacting with phosphorylated and non-phosphorylated ABI1-p65, functioning as a dominant-negative form of ABI1-p65.

Early assessment of minimal residual disease identifies patients at very high relapse risk in NPM-ALK–positive anaplastic large-cell lymphoma

AbstractDetection of minimal disseminated disease (MDD) at diagnosis correlates with relapse risk in children with anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL). We investigated whether minimal residual disease (MRD) positivity by qualitative reverse-transcriptase polymerase chain reaction (RT-PCR) for Nucleophosmin (NPM)-ALK during treatment identifies patients at the highest relapse risk. Blood and/or bone marrow of 180 patients with NPM-ALK–positive ALCL treated with Berlin-Frankfurt-Münster-type protocols were screened for NPM-ALK transcripts at diagnosis; 103 were found to be MDD-positive. MRD before the second therapy course could be evaluated in 52 MDD-positive patients. MRD positivity correlated with uncommon histology. The cumulative incidence of relapses (CIR) of 26 MDD-positive/MRD-positive patients (81% ± 8%) was significantly higher than the CIR of 26 MDD-positive/MRD-negative (31% ± 9%) and 77 MDD-negative patients (15% ± 5%) (P < .001). Five-year survival of MDD-negative and MDD-positive/MRD-negative patients was 91% ± 3% and 92% ± 5%, respectively, compared with 65% ± 9% of MDD-positive/MRD-positive patients (P < .001). Early evaluation of MRD in NPM-ALK–positive ALCL identifies patients with a very high relapse risk and inferior survival.

Abstract 3540: Evaluation of Alpha-fetoprotein (AFP), telomerase, melanoma associated antigen (MAGE1 and MAGE 3), cancer stem cell markers cytokeratin (CK) and (CD133,) as potential biomarkers for hepatocellular carcinoma (HCC).

Abstract Background. Hepatocellular carcinoma (HCC) had been recently classified as a complex disease with a wide range of risk factors. Analysis of AFP level as a tumor marker is applied in patients with a high risk of developing HCC. However, the lack of AFP sensitivity and specificity has elucidated the need for novel tumor markers. Therefore, the aim of the current study is to assess AFP, MAGE1 and MAGE 3, CK, and HCC cancer stem cell (CSC) markers (CD133, CD90, CD44) as potential biomarkers for HCC patients. Patients and Methods. This study was conducted on the peripheral blood lymphocytes (PBL) of one 130 participants involving 70 patients with HCC, thirteen patients with chronic hepatitis (CH) and 30 normal controls. For those patients, serum AFP was tested by ELISA. Telomerase, MAGE 1, and MAGE 3, CD90 and CD44 RNA expression was detected by qualitative reverse transcriptase-polymerase chain reaction (qPCR). CD133 and CK expression was measured by flowcytometry (FCM) and qPCR analysis in 10 ml of PBL of each patient. Results. All CH and HCC patients were positive for HCV antibodies. The number of cells expressing CD 133 in the patients PBL was significantly lower in HCC patients than other groups while that of CD90 and CD44 was significantly higher in HCC than in the other groups (p&amp;lt;0.01). Also, MAGE-1,MAGE-3, AFP, CD90 and CD44 expressions by qPCR were significantly higher (p&amp;lt;0.001) in HCC group than in chronic hepatitis and normal control groups. There was a highly significant difference (p&amp;lt;0.001) between groups for telomerase gene expression, which was detected in 10% and 42.3% of CHC and HCC patients respectively. The expression levels of AFP, CD44 and CD90 correlated significantly with HCC stage (r=.461, p=0.001), grade and multicentricity (r=.244, p=0.02) while CD133 showed a significant correlation with tumor size (r=.455, P=0.006) but not significantly correlated with multicentricity and HCC stage. Cellular expression of CK was significantly higher in HCC than in other groups, and showed a significant correlation with tumor size (r=.347, P=0.041) and multicentricity (r=0.513, P=0.02). Conclusion. 1) AFP, MAGE1, MAGE 3 and the number of cells expressing CK in the PBL of HCC could be used as markers for early detection of HCC. 2) The CSC markers (CD90, CD44) and AFP are highly expressed in HCC and contribute to aggressive phenotype thus they could be used as prognostic and predictive markers. Citation Format: Abdel-Rahman N. Zekri, Abeer Bahnassy. Evaluation of Alpha-fetoprotein (AFP), telomerase, melanoma associated antigen (MAGE1 and MAGE 3), cancer stem cell markers cytokeratin (CK) and (CD133,) as potential biomarkers for hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3540. doi:10.1158/1538-7445.AM2013-3540

Ring chromosome derived from BCR/ABL in an acute lymphocytic leukemia

A previously healthy 44-year-old female was referred to our hospital with an increase of peripheral white blood cell (WBC) counts and slight fever. CBC showed a hemoglobin level of 9.7 g/dL, a platelet count of 205 9 10/L, and WBC count of 128.4 9 10/L with 4 % segmented neutrophils, 5 % band form neutrophils, 4 % lymphocytes, 1 % monocytes and 86 % blasts. Bone marrow aspiration showed a hypercellular marrow (nuclear cell count: 542 9 10/L) with a massive increase of hematopoietic blasts (83.8 %). These blasts were negative for MPO and esterase. Flow cytometric analysis confirmed cells positive for CD10, CD19, and CD34, and negative for CD3, CD4, CD7, CD14, CD56, and CD8. G-banding analysis of chromosomes revealed 46XX, -9, -22, ?r1 in six of 20 metaphases (Fig. 1a) and fluorescent in situ hybridization (FISH) analyses demonstrated that the ring chromosome contained BCR/ABL fusion signals (Fig. 1b), which were detected in most metaphases (88 %). BCR/ABL transcripts were confirmed by qualitative reverse transcriptase-polymerase chain reaction and revealed minor bcr involvement. Upon these data, she was diagnosed with B lymphoblastic leukemia with t(9;22)(q34;q11.2); BCR/ABL based on the WHO classification. Ring chromosome is a circular structural chromosome abnormality that is assumed to arise from breakage and subsequent fusion of one or multiple chromosomes. This chromosomal abnormality is often unstable, leading to concomitant genetic loss or amplification. This may explain why ring chromosome is extremely rare in hematopoietic malignancies [1], although many other chromosomal abnormalities are frequently found. In this case, the FISH analysis showed multiple distinct BCR/ABL fusion signals in a ring chromosome, strongly suggesting that this ring chromosome is derived from Philadelphia chromosome t(9;22). There are several acute lymphoblastic leukemias carrying r(9)(q34q34) listed in the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, but these do not have BCR/ABL fusion [2]. This is the first case to our knowledge demonstrating the BCR/ABL fusion in a ring chromosome. It remains uncertain how this unique chromosomal structure contributes to clinical outcome. Accumulation of leukemia patients with ring chromosome linking BCR/ABL is necessary to elucidate its contribution to clinical manifestation.

Cytogenetic &amp; molecular analyses in adult chronic myelogenous leukaemia patients in north India

Background & objectives:Chronic myelogenous leukaemia (CML) is the commonest leukaemia in Asia. There is a paucity data on cytogenetic and molecular analyses of Indian CML patients. This apparently reflects the low availability of cytogenetic and molecular techniques in our country. This study aimed to document various types of BCR-ABL fusion transcripts in different phases of CML and to compare the Ph chromosome positivity/negativity vis-a-vis BCR-ABL fusion transcripts in adult CML patients.Methods:Between June 2004 and February 2009, 208 patients were diagnosed as CML in chronic phase (CP), accelerated phase (AP) and blast crisis (BC), according to standard criteria. Cytogenetic and molecular genetic analyses were performed in all patients. Various types of BCR-ABL hybrid transcripts were compared with phases of CML and cytogenetic abnormalities.Results:Among 208 CML patients, b3a2 BCR-ABL transcripts were most commonly detected (66.82%) followed by b2a2 (28.84%), b3a2 + b2a2 (3.36%), b3a2 + e19a2 (0.48%) and b2a2 + e19a2 (0.48%). b3a2 transcripts were more frequently detected than b2a2 transcripts, in the whole group of 208 as well as in 183 CML-CP patients (P<0.0001). Ph chromosome was positive in 135 of 139 patients with b3a2 transcripts and 56 of 60 patients with b2a2 transcripts, difference not being significant. Additional cytogenetic abnormalities detected in 3.8 per cent patients in CML-CP and 44 per cent patients in CML-AP/BC, did not show predilection for any BCR-ABL transcript type.Interpretation & conclusions:This study documents higher Ph positivity (96.15%) by cytogenetic analysis among CML patients, as confirmed by qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in a large patient group from north India. Both the techniques contribute towards understanding the disease biology, and have important implications for diagnosis and management of CML patients.

Basally activated nonselective cation currents regulate the resting membrane potential in human and monkey colonic smooth muscle

Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K(+), suggesting that it is regulated not only by K(+) conductances but by inward conductances such as Na(+) and/or Ca(2+). We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na(+) with equimolar tetraethylammonium or Ca(2+) with Mn(2+) inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na(+) with N-methyl-D-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (bI(NSCC)) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of bI(NSCC). In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle.

CDY1 and BOULE transcripts assessed in the same biopsy as predictive markers for successful testicular sperm retrieval

To evaluate the molecular markers CDY1 and BOULE in the same testicular biopsy for predicting success of sperm retrieval in azoospermic men. Prospective study. University-affiliated medical center. Azoospermic men (n = 92) who underwent testicular sperm extraction (TESE) and who were classified as normal spermatogenesis, mixed atrophy, spermatocyte maturation arrest, or Sertoli cell only according to their combined histological and cytological testicular findings. Quantitative and qualitative evaluation of testicular biopsies by histological and qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) expression methodologies. CDY1 and BOULE expression and the presence of sperm cells in testicular tissue. Both transcripts significantly predicted the presence of sperm cells by qualitative and quantitative methodologies. Although CDY1 had the best sensitivity by qualitative RT-PCR (98.3%), assessing both transcripts simultaneously had an additive efficacy compared with assessing CDY1 alone, improving the specificity from 84.4% to 96.3%. Assessing the expression of both CDY1 and BOULE by qualitative RT-PCR is a sensitive and feasible test for predicting the presence of sperm cells in testicular tissue and may serve as a predictive tool if repeated TESE is required.

Expression of HYAL1 and Survivin RNA as Diagnostic Molecular Markers for Bladder Cancer

Urinary tumor markers that help in the early detection of bladder cancer promise a significant improvement in sensitivity, specificity and convenience over conventional, invasive diagnostic tests. We assessed the diagnostic efficacy of hyaluronidase (HYAL1) and survivin for early bladder cancer detection. The study included 166 patients diagnosed with bladder carcinoma, 112 with benign bladder lesions and 100 healthy volunteers who served as controls. All underwent serological assessment of schistosomiasis antibody, urine cytology, and hyaluronidase (HYAL1) and survivin RNA estimation by qualitative and semiquantitative reverse transcriptase-polymerase chain reaction in urothelial cells from voided urine. Positivity rates of HYAL1 RNA and survivin RNA on qualitative reverse transcriptase-polymerase chain reaction were significantly different among the 3 groups. Mean rank using semiquantitative method was increased in the malignant vs the other groups. The best cutoff for HYAL1 and survivin RNA was 0.25 each. Using these cutoffs HYAL1 and survivin RNA sensitivity was 91% and 75%, respectively, with absolute specificity. HYAL1 RNA detected all patients with stages 0 and I bladder cancer (p <0.037). Urine cytology sensitivity improved when combined with hyaluronidase or survivin RNA on semiquantitative reverse transcriptase-polymerase chain reaction. The detection of urinary HYAL1 and survivin RNA is a promising noninvasive test for bladder cancer early detection. HYAL1 RNA was more sensitive and specific than urine cytology. Semiquantitative reverse transcriptase-polymerase chain reaction is favored for its high sensitivity and specificity.

Acute promyelocytic leukemia with insertion of PML exon 7a and partial deletion of exon 3 of RARA: a novel variant transcript related to aggressive course and not detected with real-time polymerase chain reaction analysis

We report the case of a 38-year-old man with acute promyelocytic leukemia (APL) showing a distinct breakpoint cluster region 2 (bcr2) variant transcript. Findings from bone marrow, cytogenetic, fluorescence in situ hybridization, and qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were consistent with the diagnosis of APL. Although PCR products of size 841 bp and 984 bp were amplified from bone marrow specimen, the quantitative PCR (RQ-PCR) findings were negative. Given the discrepancy in PCR results, sequencing of PCR products was performed to determine the detailed composition of these fusion transcripts. By cloning and sequencing, we discovered that these two bands were isoforms, in which one exon (exon 5, 144 bp) of the PML gene was spliced out of the smaller products (minor PCR products); one sequence (G) insertion and one base substitution (T→C) of PML exon 4 generate a stop codon in the smaller fusion transcript. In addition, a search of the Ensembl database revealed that these variant PML/RARA fusion transcripts were composed of exon 7a insertion of the PML gene and partial deletion (46 bp) of exon 3 of the RARA gene, in addition to inserted sequences of intron 7 of PML and genomic sequence ATCT of unknown origin at the fusion junction site. Although the biological significance of most atypical transcripts remains unclear, sequence analysis of these atypical products should be performed, to reveal the composition of such a fusion transcript and elucidate the molecular mechanism.

Early breast cancer patients have a higher incidence of recurrence/death from breast cancer if mammaglobin expression is detected in histologically negative axillary lymph nodes-mean 79 months follow-up

The polypeptide, Mammoglobin (MGB), has been shown to have increased expression in breast cancer. Qualitative Reverse-Transcriptase Polymerase Chain Reaction was performed to establish whether MGB was expressed in 101 patients with early breast cancer. Tumour, axillary lymph nodes, peripheral venous blood and bone marrow samples were collected between 2000-2. Tissue was also analyzed histologically. These patients have mean follow-up of 79 months. The number of reccurrence/deaths specifically attributed to breast cancer was recorded.

PML-RARalpha gene detection method optimization for quantitative PCR

Hybrid gene PML-RARalpha is the molecular target found in most cases of acute promyelocytic leukemia (APL) and has been used for diagnosis and minimal residual disease studies. The standard molecular technique employed is qualitative reverse transcriptase-polymerase chain reaction (RT-PCR), but with the emergence of real time PCR (Q-PCR), PML-RARalpha gene detection approaches have been described allowing transcript detection, with the methodological advantage of eliminating post-PCR processing. However, current protocols report the use of expensive fluorescent labeled probes, limiting its routine application in the laboratory. The objective of this study was to optimize PML-RARalpha gene detection method for Q-PCR, using SYBR® Green fluorescent dye. The analysis was performed with NB4 cellular lineage cDNA. Thermal cycling protocols, cDNA synthesis with random or specific primer and different MgCl2 and amplification primers concentrations were tested. Results show that amplification improved in the following conditions: 2 mM MgCl2, 10 pmol primers and cDNA synthesized with specific primer. There were no significant differences using annealing temperature (58°C/30 s) followed by extension (72°C/30 s) or annealing associated with extension as a single step (60°C/45 s). This paper demonstrates the optimization of PML-RARalpha gene detection for Q-PCR studies using a technique considered sensitive and less expensive for routine use in the laboratory.

Identification of Leptin Gene Expression in Sinusoidal Endothelial Rat Liver Cells

Sinusoidal endothelial liver cells (SECs) have a key role in the pathophysiology of chronic liver disease. Leptin is an important profibrogenic and proinflammatory cytokine whose expression in sinusoidal endothelial liver has not been documented. The authors studied the potential of rat SECs to express the leptin and leptin receptor genes. Two cell lines of rat SECs were generated from a male rat liver by pronase-collagenase perfusion and dilution cloning. They were characterized according to morphology, ploidy, von Willebrand antigen immunoreactivity, CD31 transcription, matrix metalloproteinase secretion, and pseudocapillary formation. Expression of the leptin and leptin receptor genes was studied using qualitative reverse transcriptase-polymerase chain reaction. Both cell lines fulfilled the accepted criteria for consideration as being derived from the liver sinusoidal endothelium. Confluent monolayers of both cell lines transcribed leptin and leptin receptor genes. This work demonstrated that SECs can transcribe the leptin gene in vitro, cotranscribing with the leptin receptor gene. Leptin production and signaling at this level could be of paramount importance in liver physiopathology; further studies of this issue are warranted because it represents a potential intervention point during chronic liver diseases.

Differential expressions of heme oxygenase-1 gene in CD25 − and CD25 + subsets of human CD4 + T cells

Growing evidence suggests that the immunomodulatory heme oxygenase-1 (HO-1) may have an important role in regulating T-cell responses. In this study, we investigated whether CD4 +CD25 − and CD4 +CD25 + T cells of human CD4 + subpopulation could differentially express HO-1. Our results obtained from qualitative reverse transcriptase-polymerase chain reaction and quantitative flow cytometry analyses revealed that the CD4 +CD25 + T cells constitutively express HO-1 and that T cell stimulation with plate-bound anti-CD3 in combination with soluble anti-CD28 not only induced HO-1 gene expression in the CD4 +CD25 − T cells but also up-regulated HO-1 gene expression in the CD4 +CD25 + T cells. Our further studies showed that CD28 signal alone was enough to induce HO-1 expression and CD3 signal, of which signal alone did not induce HO-1 expression, was required at least for full HO-1 expression in both CD25 − and CD25 + subsets of human CD4 + T cells. In addition, transfection of human Jurkat T cells with HO-1 suppressed the cellular proliferation, and this effect was reversed by zinc protoporphyrin, a specific HO competitive inhibitor. Taken together, we have first reported that human CD4 +CD25 + regulatory T cells constitutively express HO-1 and that HO-1 inhibits Jurkat T cell proliferation.

Lack of seronegative hepatitis C virus infections in patients with chronic renal failure.

Recent reports have indicated that serologic testing for hepatitis C virus (HCV) in patients with chronic renal failure may be inadequate to detect infections in this patient population. We prospectively tested 258 patients with end-stage renal disease who were referred for evaluation for renal transplantation for anti-HCV using a second-generation enzyme immunoassay (EIA) and a second-generation qualitative reverse-transcriptase polymerase chain reaction (RT-PCR). We confirmed all positive EIAs with a third-generation recombinant immunoblot assay and genotyped RT-PCR-positive specimens. We found that 22 patients (8.5%) had serological evidence of HCV infection. Nineteen (86%) of the antibody-positive patients were viremic (HCV RNA positive). All but 1 of the patients was infected with HCV genotype 1. None of the 233 HCV antibody-negative patients were shown to be viremic by RT-PCR. No additional HCV cases were detected by screening all patients for HCV RNA by RT-PCR. However, RT-PCR is a valuable adjunct to serology in antibody-positive patients to distinguish resolved from active infections.

Intrauterine expression of prothrombin in the sprague-dawley rat.

Thrombin appears to underlie myometrial contractions in response to intrauterine bleeding. In a similar fashion, thrombin generated within the uterus in the absence of active bleeding could also produce contractions. These studies sought to determine whether functionally active prothrombin is expressed in the pregnant and nonpregnant rat uterus. Uteri were obtained from proestrus/estrus and timed-pregnant Sprague-Dawley rats. Western blots were performed using antithrombin antibodies. Immunohistochemical studies were performed using the same antibodies along with the Vector Elite ABC kit. Qualitative reverse transcriptase-polymerase chain reaction studies were performed using rat prothrombin-specific oligonucleotide primers. In vitro uterine contraction studies were performed using Taipan snake venom (an exogenous prothrombinase) and components of the plasma prothrombinase complex (Factors Xa and V) with and without pretreatment with thrombin inhibitors (heparin or hirudin). The Western blots demonstrated prothrombin peptides in myometrial tissue from estrus and pregnant rats. The immunohistochemical studies confirmed prothrombin peptides in both the circular and longitudinal myometrium, along with the endometrium. The reverse transcriptase-polymerase chain reaction studies demonstrated prothrombin mRNA in the endometrium and placenta, but not in the myometrial smooth muscle. The Taipan snake venom stimulated a significant increase in contractions, which were suppressed by pretreatment with heparin and hirudin. The Factor Xa and V complex also significantly stimulated uterine contractions, which were likewise inhibited by hirudin. These studies provide evidence supporting the expression of functionally active prothrombin in the pregnant and nonpregnant rat uterus. Based on the presence of its mRNA, prothrombin appears to be synthesized in the endometrium and placenta; in contrast, the myometrial smooth-muscle cells appear to sequester preformed prothrombin. These results support the hypothesis that intrauterine thrombin could play an autocrine/paracrine role in the regulation of contractile activity.

Effective Management of Hepatitis C Molecular Testing Improves Test Use Without Compromising Patient Management

The availability of effective antiviral therapy for hepatitis C has increased the need for molecular detection and quantification of circulating hepatitis C viral particles. The limits of detection differ for the quantitative and qualitative reverse transcriptase polymerase chain reaction (RT-PCR) assays; furthermore, adequate patient assessment requires both detection of hepatitis C virus when it is present and quantitation of the viral load when possible. The combination of these factors promotes the simultaneous ordering of both tests with the possibility of generating redundant test information. To reduce the number of unnecessary hepatitis C tests performed. We established a reflexive testing protocol for quantitative and qualitative RT-PCR testing for hepatitis C. During a 3(1/2)-month interval, 170 qualitative RT-PCR hepatitis C tests were eliminated (a 59.4% reduction in the number of these tests). This reduction was achieved without a clinically significant change in turnaround time or a compromise of patient care. Establishing the quantitative and qualitative RT-PCR tests in-house and adopting the reflexive testing protocol was cost-effective and did not compromise patient management or care.

Early detection of BCR-ABL transcripts by quantitative reverse transcriptase–polymerase chain reaction predicts outcome after allogeneic stem cell transplantation for chronic myeloid leukemia

The reverse transcriptase-polymerase chain reaction (RT-PCR) has become widely used for monitoring minimal residual disease after allogeneic stem cell transplantation (SCT) for chronic myeloid leukemia (CML). However, most of these studies were performed using qualitative RT-PCR, and the interpretation of the results obtained has been conflicting. The correlation of a quantitative RT-PCR test performed early after SCT (at 3 to 5 months) and long-term outcome of CML patients surviving for more than 6 months was studied. Between January 1991 and June 1999, data from 138 CML patients who received allografts were evaluated. Early RT-PCR results were classified as (1) negative if there were no BCR-ABL transcripts detected (n = 61), (2) positive at low level if the total number of BCR-ABL transcripts was less than 100 per microg RNA and/or the BCR-ABL/ABL ratio was less than 0.02% (n = 14), or (3) positive at high level if transcript levels exceeded the thresholds defined above (n = 63). Three years after SCT the cumulative incidence of relapse was 16.7%, 42.9%, and 86.4%, respectively (P =.0001). The relationship between BCR-ABL transcript level and probability of relapse was apparent whether patients had received sibling or unrelated donor SCT and also whether or not the transplantation was T cell depleted. The results suggest that quantitative RT-PCR performed early after SCT is useful for predicting outcome and may help to define the need for further treatment.

Expression of tissue inhibitor of metalloproteinase-1, -2, and -3 during neointima formation in organ cultures of human saphenous vein.

Degradation of the extracellular basement membrane is implicated in atherosclerosis, restenosis after angioplasty, and intimal thickening of vein grafts. Upregulation of metalloproteinase (MMP)-2 and MMP-9 accompanies neointima formation in cholesterol-fed rabbits, in rat and pig models of angioplasty, and in organ cultures of human saphenous veins. MMPs are inhibited by binding to tissue inhibitors of MMPs (TIMPs). Relatively little is known about their regulation in relationship to neointima formation; thus, we investigated TIMP expression in the organ culture model. Qualitative reverse transcriptase-polymerase chain reaction of mRNA extracted from veins showed that TIMP-1, TIMP-2, and TIMP-3 are each expressed before and after culture. Zymography revealed that TIMP-1 was the most abundant TIMP secreted and that its secretion increased dramatically between 0 to 2 and 12 to 14 days of culture. An enzyme-linked immunosorbent assay showed that TIMP-1 secretion increased from 3.2+/-1.5 (mean+/-SE) to 32+/-6 ng/mg wet weight per day (n=5, P<0.01). Immunocytochemical testing localized the increased expression of TIMP-1 to neointimal smooth muscle cells. Although less abundant, TIMP-2 secretion also increased from 0.8+/-0. 3 to 4.7+/-0.2 ng/mg wet weight per day (n=5, P<0.001), and tissue levels increased from 33+/-7 to 150+/-70 ng/mg wet weight (P<0.05). TIMP-2 was also immunolocalized to neointimal smooth muscle cells and their surrounding matrix. TIMP-3 was not secreted but was detected variably and constitutively in tissue extracts (160+/-120 and 170+/-100 ng/mg wet weight [n=9] on days 2 and 14, respectively). TIMP-3 was found in the cells and extracellular matrix of the media and adventitia before culture and to a lesser extent in the neointima after 14 days of culture. Rates of total TIMP secretion on day 14 exceeded those of MMP-2 and MMP-9 (10.6+/-1.9 and 15.6+/-2.3 ng/mg wet weight per day, respectively). Consistent with this, in situ zymography showed that MMP gelatinase activity was highly localized to cell bodies in the media and neointima. Secretion of TIMP-1 and TIMP-2 is greatly increased during neointima formation in human saphenous veins. TIMP-1 is readily released, whereas TIMP-2 remains partially attached and TIMP-3 exclusively attached to the extracellular matrix. Regulation of TIMP expression is therefore an important determinant of net MMP activity during neointima formation, restricting it to the pericellular environment.

Intragraft cytokine expression in tolerant rat renal allografts with rapamycin and cyclosporin immunosuppression.

The Th-1/Th-2 paradigm proposes clonal expansion of Th-2 lymphocytes as the basis of tolerance towards allografts. Intragraft cytokine expression was evaluated in a highly stringent model of renal transplantation. ACI and Lewis rats were used as donors and recipients, respectively, for heterotopic renal transplantation. Group A (n = 8) received a single dose of rapamycin and cyclosporin 12 h prior to engraftment, followed by 7 d of cyclosporin post-operatively. Isografts (Group B, n = 5) and control allografts (Group C, n = 4) received no immunosuppression. Sacrifice was performed after 120 d. Intragraft expression of IL-10, IL-4, and IFN-gamma was determined using qualitative reverse transcriptase-polymerase chain reaction (RT-PCR). All groups had functionally normal grafts at sacrifice, with 50% histological tolerance among Group A animals. No isografts showed evidence of cellular infiltrate, and all control allografts showed severe rejection. IL-10 was only detected in the tolerant animals (p < 0.001). Similarly, IL-4 was detected predominantly in the tolerant allografts (p < 0.05). IFN-gamma was only isolated in rejected allografts, whether treated or untreated (p < 0.001). We conclude that the expansion of Th-2 cells is associated with tolerance, while the expansion of Th-1 cell is associated with acute cellular rejection.