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Biomedical subjects

A Raza

Publications and source records attributed to A Raza.

At least 19 recordsLinked to original sources
PubMed1999

Correlation of tumor necrosis factor alpha (TNF alpha) with high Caspase 3-like activity in myelodysplastic syndromes.

Increased intramedullary apoptotic death of hematopoietic cells is thought to contribute to the ineffective hematopoiesis in myelodysplastic syndromes (MDS). Furthermore, high amounts of tumor necrosis factor alpha (TNF alpha) have previously been correlated with apoptosis in MDS marrows. The present studies were undertaken to examine the status of two key downstream effectors of TNF alpha signaling, i.e. Caspase 1 and Caspase 3 enzymes, using a fluorometric assay in the bone marrow aspirate mononuclear cells (BMMNC) in relation to apoptotic DNA fragmentation detected by in situ end-labeling (ISEL) of DNA and with localization of TNF alpha in the corresponding biopsies from 14 MDS patients. Both Caspase 1 and 3 were detectable in freshly harvested BMMNC, albeit median Caspase 3 levels (47.5 units/mg protein) being almost 10 times higher than Caspase 1 (4.0 units/mg protein). Upon short-term culture for 4 h in a serum-supplemented medium in vitro a significant increase was seen in Caspase 3 activity (58.8 +/- 13.9 at 0 h vs. 177.8 +/- 55.2 units/mg protein at 4 h, n = 14, P = 0.017) and in percent cells labeled by ISEL (apoptotic index or AI%: 0.76% +/- 0.25% vs. 3.99% +/- 1.1%, n = 14, P = 0.004, respectively). Caspase 1 activity increased after 15 min in culture. Interestingly, TNF alpha levels measured by immunohistochemistry correlated with the net increase in Caspase 3 activity after 4 h (p = 0.517, n = 13, P = 0.07) and the starting levels of Caspase 1 at 0 h correlated with the Caspase 3 levels attained at 4 h (p = 0.593, n = 13, P = 0.033). Additionally when TNF alpha-positive bone marrows (8/14) were compared with the negative marrows (6/14) the Caspase 3 levels were significantly higher in the TNF alpha-positive marrows (189.6 +/- 66.2 vs. 25.0 +/- 14.6 units/mg protein, respectively, P = 0.043). The increase in AI%, though not statistically significant, was also higher in the TNF alpha-positive marrows. Finally in HL60 cells the effects of different Caspase inhibitors and pentoxifylline (PTX) (interferes with lipid signaling of cytokines) on TNF alpha-induced apoptosis were evaluated. TNF alpha treatment significantly increased AI% (P < 0.003) as compared to the untreated controls. A co-treatment with three Caspase inhibitors, zVAD.FMK (inhibitor of Caspases 1 and 3, 10 microM/l), Ac.YVAD.FMK (Caspase 1 inhibitor, 1 microM/l), Ac.DEVD.FMK (Caspase 3 inhibitor, 10 microM/l) as well as PTX (250 microM/l) significantly curtailed the AI% induced by TNF alpha. The present studies thus identify the downstream effectors of TNF alpha-inducible apoptosis in MDS and so also the suppressors of TNF alpha apoptotic signaling. These results may have significant clinical implications in the therapy of MDS in the future.

Apoptosis↗
PubMed1999

Evidence for involvement of tumor necrosis factor-alpha in apoptotic death of bone marrow cells in myelodysplastic syndromes.

We previously reported excessive apoptosis and high levels of tumor necrosis factor-alpha (TNF-alpha) in the bone marrows of patients with myelodysplastic syndromes (MDS), using histochemical techniques. The present studies provide further circumstantial evidence for the involvement of TNF-alpha in apoptotic death of the marrow cells in MDS. Using our newly developed in situ double-labeling technique that sequentially employs DNA polymerase (DNA Pol) followed by terminal deoxynucleotidyl transferase (TdT) to label cells undergoing apoptosis, we have characterized DNA fragmentation patterns during spontaneous apoptosis in MDS bone marrow and in HL60 cells treated with TNF-alpha or etoposide (VP16). Clear DNA laddering detected by gel electrophoresis in MDS samples confirmed the unique length of apoptotic DNA fragments (180-200 bp). Surprisingly, however, phenotypically heterogeneous population of MDS cells as well as the homogenous population of HL60 cells showed three distinct labeling patterns after double labeling--only DNA-Pol reaction, only TdT reaction, and a combined DNA Pol + TdT reaction, albeit in different cohorts of cells. Each labeling pattern was found at all morphological stages of apoptosis. MDS mononuclear cells, during spontaneous apoptosis in 4 hr cultures, showed highest increase in double-labeled cells (DNA Pol + TdT reaction). Interestingly, this was paralleled by TNF-alpha-induced apoptosis in HL60 cells. In contrast, VP16 treatment of HL60 cells led to increased apoptosis in cells showing only TdT reaction. The double-labeling technique was applied to normal bone marrow and peripheral blood mononuclear cells after treatment with known endonucleases that specifically cause 3' recessed (BamHI), 5' recessed (PstI), or blunt ended (DraI) double-stranded DNA breaks. It was found that the DNA-Pol reaction in MDS and HL60 cells corresponds to 3' recessed DNA fragments, the TdT reaction to 5' recessed and/or blunt ended fragments, and a combined "DNA Pol + TdT reaction" corresponds to a copresence of 3' recessed with 5' recessed and/or blunt ended fragments. Clearly, therefore, apoptotic DNA fragments, in spite of a unique length, may have differently staggered ends that could be cell (or tissue) specific and be selectively triggered by different inducers of apoptosis. The presence of TNF-alpha-inducible apoptotic DNA fragmentation pattern in MDS supports its involvement in these disorders and suggests that anti-TNF-alpha (or anticytokine) therapy may be of special benefit to MDS patients, where no definitive treatment is yet available.

Apoptosis↗
PubMed1999

Biologic characteristics of patients with hypocellular myelodysplastic syndromes.

Rates of proliferation and apoptosis as well as expression of tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta) and the number of macrophages were measured in bone marrow (BM) biopsies of 33 patients who presented with hypocellular (cellularity < 30%) myelodysplastic syndromes (MDS). Results showed that 2/3 of the patients had high apoptosis, high cytokine levels and large number of macrophages in their biopsies while 1/3 did not. Apoptosis and TNF-alpha levels were directly related (r = 0.583, P = 0.003, n = 24) as was apoptosis and the degree of anemia (P = 0.033, n = 18). A subgroup of patients with abnormalities of chromosomes 5 or 7 had higher platelets (P = 0.026) and higher apoptosis (P = 0.038) when compared with the rest of the group. Eight patients had no evidence of apoptosis and almost no detectable TNF-alpha in their biopsies. We conclude that within the hypocellular variant of MDS, there may be two distinct sub-groups of patients, one who present with high cytokine-mediated intramedullary apoptosis and the other who may be better characterized as having a stem-cell failure defect since they showed no evidence of apoptosis.

Adolescent↗
PubMed1999

The effects of EMLA and a topical formulation of 4% amethocaine (Ametop) on pain associated with retrobulbar injection.

Retrobulbar block is commonly performed to provide anaesthesia for cataract extraction. This technique can cause significant discomfort. A prospective, randomised, placebo-controlled trial was carried out to investigate the efficacy of a eutectic mixture of local anaesthetics (EMLA) and a 4% amethocaine topical formulation (Ametop) in reducing the pain of retrobulbar injection. Ametop and EMLA proved to be of similar efficacy, both being superior to a placebo in alleviating the discomfort of retrobulbar block. No significant side-effects were observed with the use of either formulation.

Aged↗
PubMed1999

Extensive apoptosis of bone marrow cells as evaluated by the in situ end-labelling (ISEL) technique may be the basis for ineffective haematopoiesis in patients with myelodysplastic syndromes.

Apoptosis is a gene-directed cellular self-destruction which begins with internucleosomal cleavage of DNA and ends eventually with fragmentation of the nucleus. We have shown that the technique of ISEL of fragmented DNA appears to be an accurate and reliable measurement of the early stages of apoptosis. The present study was undertaken in order to define the incidence of programmed cell death in bone marrow (BM) haematopoietic and stromal cells of myelodysplastic syndromes (MDS). The ISEL technique was employed in 21 BM biopsies of MDS patients. The analysis showed that in 11/21 patients, >70% cells (high score) were undergoing programmed cell death while 5 patients showed up to 1/3 of the biopsy containing apoptotic cells and 2 patients had only few occasional ISEL positive cells. Stromal cells including fat cells, endothelial cells and fibroblasts were frequently in apoptosis in large clusters. Our results indicate that extensive apoptosis of haematopoietic cells documented in BM biopsies of MDS patients may be the explanation for the ineffective haematopoiesis which is the hallmark of these disorders.

Aged↗
PubMed1999

Biological characteristics of myelodysplastic syndrome patients who demonstrated high versus no intramedullary apoptosis.

Spontaneous intramedullary apoptosis was measured in bone marrow (BM) biopsies of 175 patients with myelodysplastic syndromes (MDS) using in situ end-labeling (ISEL) of fragmented DNA. Two groups of high (n=71) versus low (n =43) levels of apoptosis were identified while 61 patients were ISEL-negative. Semiquantitative assessment of 3 cytokines, the number of macrophages and in vivo labeling indices (LI) were also determined from consecutive sections of the biopsy. Patients with high apoptosis levels tended to have a high LI (p=0.013), more macrophages in their BM biopsies (p=0.006) and higher tumor necrosis factor alpha (TNF-alpha) levels (not significant) compared to patients with no apoptosis. In addition, low risk MDS patients had significantly lower rates of apoptosis (p = 0.047) and lower levels of TNF-alpha (p = 0.055) compared to high-risk MDS patients. We conclude that the genesis of cytopenias in MDS is of multifactorial origin and that cytokine-associated apoptosis clearly identifies a distinct biological subgroup of patients who may benefit selectively by use of anti-cytokine therapies.

Aged↗
PubMed1999

Biologic characteristics of 164 patients with myelodysplastic syndromes.

Rates of proliferation, apoptosis and cytokine expression were measured in bone marrow (BM) biopsies of 164 myelodysplastic syndrome (MDS) patients. There were 107 males and 57 females. Median age was 69 years and 101 had refractory anemia (RA), 17 RA with ringed sideroblasts (RARS), 38 with RA and excess blasts (RAEB) and 8 with RAEB in transformation (RAEB-t). Apoptosis measured by in-situ end labeling (ISEL) was directly related to the number of macrophages (p = 0.028, n = 83). Mean tumor necrosis factor alpha (TNF-alpha) and ISEL positivity were higher in RAEB + RAEB-t patients (p = 0.0554 and p = 0.06 respectively) while hemoglobin was higher for RA + RARS group (p = 0.0472). Patients with high apoptosis had lower white blood cell counts (p = 0.0009), lower percentage of blasts (p = 0.0009) and higher number of macrophages (p = 0.0086). We conclude that measurements of apoptosis, proliferation and cytokine expression provide important biological information which helps to distinguish RA + RARS patients from RAEB + RAEB-t patients, and may be of additive prognostic significance.

Adolescent↗
PubMed1999

The relative extent and propensity of CD34+ vs. CD34- cells to undergo apoptosis in myelodysplastic marrows.

The paradox of peripheral cytopenias despite cellular bone marrow (BM) observed in myelodysplastic syndromes (MDS) has been associated with excessive intramedullary apoptosis of hematopoietic cells. Since MDS is regarded as a stem cell disorder, the present studies were undertaken to examine the relative susceptibility and propensity of early progenitor CD34+ cells to undergo apoptosis as compared to more maturing/matured CD34- cells. Five serial studies were performed on 4 independent groups of 36 newly diagnosed MDS patients. First, in 2 separate groups of 16 and 8 patients each, measurement of the extent of apoptosis in CD34+ and CD34- fractions of the BM aspirate mononuclear cells was carried out using independent biparametric flow cytometry methods, CD34 labeling/terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (n = 16), and CD34 labeling/reduced uptake of nucleic acid staining dye LDS751 (n = 8). The difference in the median degrees of apoptosis in CD34+ vs. CD34- cells was not statistically significant by either technique (P = 0.583 and P = 0.674 for TUNEL and LDS751, respectively). In the next group of 4 MDS patients, a double-labeling was performed on plastic embedded marrow biopsy sections, to detect CD34 antigen with specific monoclonal antibody and apoptosis by in situ end labeling (ISEL) of fragmented DNA. Despite high overall apoptosis (56.2% +/- 18.4%), only an occasional CD34+ cell was found to be simultaneously labeled with ISEL. Finally, in the last group of 8 MDS patients, CD34+ cells were separated from CD34- cells on affinity column and cultured in serum containing medium for 4 hours. At 0- and 4-hour time points, ISEL was carried out to label apoptotic cells. In addition, a fluorometric assay was employed to estimate the activity of a proapoptotic enzyme, Caspase 3. Both the net increase in % ISEL labeled cells (apoptotic index or AI) and Caspase-3 activity were significantly lower in CD34+ cells as compared to CD34- cells (AI, 0.87% +/- 0.5% vs. 3.97% +/- 1.4%, n = 6, P = 0.028 and Caspase-3 Units/mg protein, 46.9 +/- 25.0 vs. 71.7 +/- 23.03, n = 5, P = 0.042, respectively). We conclude that when estimated in a total population of mononuclear cells, CD34+ cells and CD34- cells show comparable degrees of apoptosis. However, once separated the CD34+ fraction demonstrates lower propensity to undergo apoptosis, thereby suggesting the CD34- fraction as being a possible source for proapoptotic signaling.

Aged↗
PubMed1998

Initial transforming event in myelodysplastic syndromes may be viral: case for cytomegalovirus.

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders which begin in a pluripotential bone marrow (BM) stem cell. This early stem cell is believed to acquire a growth advantage over its neighbors as a result of an initial transforming event, the nature of which has remained obscure. In this paper, we propose that pathogens such as those belonging to the herpesvirus family of DNA viruses may play a role in the initial transformation of the stem cell. The case for cytomegalovirus (CMV) as a representative of this family of viruses is discussed at length and a molecular mechanism which may be involved in the oncogenic activity of CMV is proposed. No proof has been presented to implicate CMV directly in MDS, but circumstantial evidence which supports such a possibility is provided.

Apoptosis↗
PubMed1998

Hypothesis: myelodysplastic syndromes may have a viral etiology.

An 'initial transforming event(s)' in a pluripotential bone marrow (BM) stem cell confers a growth advantage upon it leading to clonal expansion accompanied by dysplastic maturation resulting in myelodysplastic syndromes (MDS). The nature of this 'initial' event in MDS is obscure. We propose that MDS can begin as a viral disease. It may be a dormant lentivirus which is made oncogenic by 'promoting events' such as immunosuppression, or a second viral infection. The infected cell may not be a BM stem cell, but a cell belonging to the BM stroma or to the immune system. Dysregulated cytokine production as a consequence of the infection can change the BM microenvironment in such a way that optimal growth support is provided only to a rapidly proliferating stem cell. Karyotypically marked (or unmarked) abnormal stem cells may exist or arise frequently but do not thrive in a 'normal' cytokine milieu. However, with the changed BM landscape, these abnormal clones may enjoy a growth advantage leading to a monoclonal hypercellular BM and variable cytopenias. Circumstantial evidence to support the possibility that the initial transforming event in MDS is a viral insult is presented in this hypothesis paper.

Cell Transformation, Viral↗
PubMed1998

Apparent expansion of CD34+ cells during the evolution of myelodysplastic syndromes to acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are highly proliferative bone marrow (BM) disorders where the primary lesion presumably affects a CD34+ early progenitor or stem cell. We investigated the proliferative characteristics of CD34+ cells of 33 untreated MDS patients (19 RA, 5 RARS, 7 RAEB, 2 RAEBt) and five patients with acute myeloid leukemia after MDS (sAML). All patients received a 1-h infusion of the thymidine analogue iodoor bromodeoxyuridine intravenously before a BM aspirate and biopsy was taken. A double-labeling immunohistochemistry technique by monoclonal anti-CD34 (QBend/10) and anti-IUdR/BrdU antibodies was developed and performed. By this technique we recognised CD34+ and CD34- cells actively engaged in DNA synthesis or not. As MDS evolves a significant increase occurred in the percentage of CD34+ cells of all myeloid cells (mean value: RA/RARS 1.67%; RAEB(t) 8.68%; sAML 23.83%) as well as in the percentage of proliferating CD34+ cells of all myeloid cells (RA/RARS 0.19%; RAEB(t) 0.43%; and sAML 3.30%). This was associated with a decreasing trend in the overall myeloid labeling index (LI: RA/RARS 25.8%, RAEB(t) 24.6% and sAML 21.5%). This decrease in overall myeloid LI is due to an exponential increase in the proportion of CD34+ cells of the proliferating compartment during MDS evolution (RA/RARS 0.35%, RAEB(t) 1.44% and sAML 11.98% of all S-phase cells). These CD34+ cells appeared to proliferate more slowly than their more mature CD34 negative counterparts, since we found a progressive increment in the mean total cell cycling time (Tc) of all myeloid cells during MDS progression (RA/RARS 39.8, RAEB(t) 45.2 and sAML 65.8 h). This study showed that during MDS evolution to sAML the CD34+ compartment develops a growth advantage leading to apparent expansion.

Acute Disease↗
PubMed1998

Endobrst: simple and safe is best.

Minimal-access treatment for ureteropelvic junction (UPJ) obstruction is becoming increasingly complex. Is there still a place for the simple procedure of balloon dilatation? To examine the symptomatic and renographic results of patients at least 6 months after balloon disruption of an obstructed UPJ, we carried out a prospective audit of outcome as judged by DTPA renograms and clinical follow-up. The procedure has been performed on a total of 21 renal units (20 patients) with follow-up of 6 to 30 months (mean 22 months). Of these, 18 (86%) were asymptomatic at 3 months, although this success rate dropped to 17 (81%) by 6 months. Renographic split function improved in 11 renal units, and excretion improve in 14. Perioperative complications were few and associated with stent insertion. Three of the patients in whom the procedure failed went on to nephrectomy (14%); all had poor renal function (split <20%) at presentation. Although the quoted success rates are lower for endoluminal balloon rupture of stenosis treatment than open pyeloplasty, the former technique has significantly less morbidity. Our nephrectomy rate reflects our less than ideal early patient selection, and our balloon dilation technique is not recommended as a salvage procedure for patients in whom nephrectomy is inevitable. The learning curve is quick to climb, and this is a fundamentally simple procedure with medium-term results that remain encouraging.

Adult↗
PubMed1998

Tumor necrosis factor-alpha levels decrease with anticytokine therapy in patients with myelodysplastic syndromes.

Tumor necrosis factor-alpha (TNF-alpha) levels were measured in the serum (sTNF-alpha) or bone marrow (BM) biopsies of 43 patients with myelodysplastic syndromes (MDS) who subsequently received therapy with a combination of pentoxifylline and ciprofloxacin (PC) with or without dexamethasone (PCD). All 43 patients received only PC therapy for 12 weeks, after which 18 of 36 nonresponders received PCD. A total of 18 of 43 patients showed a hematologic or cytogenetic response or both. BM TNF-alpha levels were semiquantitatively assessed using immunohistochemistry on a scale of 0-8+ and in the serum using enzyme linked immunoassay. The median TNF-alpha for the entire group was 3.0 in BM and 6.9 pg/ml in the serum, and 14 patients had no detectable levels. Responders had higher BM levels (median 3.5 vs. 2.0) than nonresponders, although this was not statistically significant. During PC therapy, a decline in BM TNF-alpha level was seen in the entire group, which was significant at 2 weeks (p = 0.02), 8 weeks (p = 0.001), and 12 weeks (p = 0.0001). Both responders (p = 0.01) and nonresponders (p = 0.03) had a decline at 8 weeks, but at 12 weeks, only the responders continued to show a significant decline (p = 0.03). We conclude that MDS patients with high BM TNF-alpa levels have a better chance of responding to PCD therapy and that the therapy is quite successful in reducing the TNF-alpha levels in a sustained fashion. Future studies need to be directed at identifying agents that would be more potent suppressors of the proapoptotic cytokines in these patients.

Aged↗
PubMed1997

Detection of hyperdiploid malignant cells in body cavity effusions by fluoresence in situ hybridization on ThinPrep slides.

BACKGROUND: Benign body cavity effusions sometimes cannot be distinguished from malignant ones by conventional cytology. The authors performed fluorescence in situ hybridization (FISH) on ThinPrep slides using chromosome specific probes to see if hyperdiploid malignant cells could be detected in 20 body cavity effusions. The results were then compared with those of conventional cytology. METHODS: A total of 20 body cavity effusions from 19 patients were studied using conventional cytology and FISH. Probes specific for chromosomes 3, 8, 10, and 12 were used to detect hyperdiploidy on ThinPrep slides (Cytyc Corporation, Boxborough, MA). RESULTS: A total of 13 patients had malignant conditions (either prior history of malignancy or the presence of malignancy anywhere in the body). Conventional cytology and FISH were both positive in 5 of these patients (6 samples) and negative in 2 patients. The results for one sample were inconclusive by both methods. There were 5 discrepant cytology-FISH results in patients with malignant conditions. One sample was positive by FISH and negative by cytology, one was positive by FISH and "atypical" by cytology, and three were inconclusive by FISH and negative by cytology. FISH results were either negative (in 4 samples) or inconclusive (in 2 samples) in the 6 patients with benign conditions. CONCLUSIONS: FISH can detect hyperdiploid malignant cells in body cavity effusions and is especially useful when the major cell population consists of malignant cells that cannot be differentiated from mesothelial or "atypical" cells. It is less useful in detecting a small population of malignant cells hidden in an inflammatory or reactive cell background. More studies are needed to establish diagnostic criteria further and to assess the clinical usefulness of this procedure.

Adult↗
PubMed1997

Local anesthesia for fine-needle aspiration biopsy of palpable breast masses: the effectiveness of a jet injection system.

To determine the effectiveness of the Biojector 2000 needle-free lidocaine injection system in achieving satisfactory local anesthesia for fine-needle aspiration (FNA) of palpable breast lesions, we studied 29 female patients. Each patient served as her own control and had two FNA biopsies performed on the lesion. The first FNA biopsy was preceded by either no anesthesia, ethyl chloride cold spray, or traditional needle lidocaine injection. The second FNA was preceded by the Biojector 2000. Twenty-four patients (83%) reported that they preferred the Biojector 2000 over either no anesthesia, ethyl chloride spray, or needle and syringe lidocaine injection. The Biojector 2000 needle-free injection system is an effective and useful method of local anesthesia for FNA of palpable breast masses.

Adult↗
PubMed1997

Cytokines, molecular biological abnormalities, and acute myelogenous leukemia.

Leukemia cell proliferation is dependent upon cytokines produced by the leukemia cells or by the microenvironment under stimulation by leukemia cells. Abnormal expression of the p53, rb, and ras genes is associated with cytokine production, suggesting that abnormal expression can affect leukemia cells by affecting intracellular growth controls and by stimulating cytokine production. While these observations suggest that cytokines can be used to stimulate leukemia proliferation during cytotoxic therapy increasing the sensitivity to treatment, they also suggest that inhibition of cytokine affects could increase clinical responses by reducing leukemia cell regrowth between courses of therapy.

Animals↗