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Biomedical subjects

J K Prasad

Publications and source records attributed to J K Prasad.

At least 19 recordsLinked to original sources
PubMed2000

Extracellular Ca(2+) suppresses endotoxin-inducible tissue factor activation in monocytic THP-1 cells.

BACKGROUND: Monocytic tissue factor (TF), an initiator of extrinsic blood coagulation, is often activated under various inflammatory conditions including endotoxemia. This activation could be a contributing factor to the manifestation of disseminated intravascular coagulation following septic shock. HYPOTHESIS: We herein determine if extracellular Ca(2+) ([Ca(2+)](ex)) regulates bacterial endotoxin (LPS)-inducible monocytic TF activation. METHODS: We have employed a model monocytic cell line (THP-1) to explore the mode of action of [Ca(2+)](ex) on the modulation of LPS-induced TF activation. TF activity was measured by a single stage clotting assay, while TF expression as well as LPS recognition and its receptor expression were studied in immunofluorescent approaches. RESULTS: LPS-induced TF activation was inversely correlated to [Ca(2+)](ex). Upon exposure of THP-1 cells to LPS (1.5 microg ml(-1)) for 6 h in the Hanks' medium without CaCl(2), TF was activated by nearly 10-fold. TF activation appreciably decreased with the increasing [Ca(2+)](ex). No more than 3.5-fold TF activation was detected at 5 mM [Ca(2+)](ex). Consistent with the significantly lower degree of TF activation, LPS-induced TF expression at 5 mM [Ca(2+)](ex) was 60 per cent less than that without [Ca(2+)](ex). FACScan analysis showed that LPS recognition was significantly blocked at 5 mM [Ca(2+)](ex) which however had no effect on the expression of CD14 and CD11b, the proposed major LPS receptors. Moreover, LPS binding in vitro was significantly inhibited by 5 mM CaCl(2). CONCLUSION: Our results demonstrate that [Ca(2+)](ex) blocked LPS recognition without affecting its receptor expression on THP-1 monocytes. This insensitivity to LPS thereby resulted in the depressed inducible monocytic TF expression and activation.

Antigens, CD↗
PubMed1999

I. Suppression by compound 48/80 of bacterial endotoxin-inducible monocytic tissue factor activity: direct blockade of factor VII binding to THP-1 monocytes.

Hypercoagulation with upregulated monocytic tissue factor (TF) activity often occurs under a variety of inflammatory conditions including endotoxemia. The antagonism to bacterial endotoxin (LPS) signaling often results in the depression in TF upregulation. We herein report that compound 48/80 (48/80) significantly depressed LPS-induced TF activity in human and cebus monkey peripheral blood monocytes. Employing a model monocyte-like cell line (THP-1), we explored the regulatory mechanism to identify the inhibitory site(s) of 48/80. We determine whether the inhibition results from the blockade of LPS signaling. 48/80 dose-dependently inhibited LPS-induced TF activity. Chase of LPS-challenged cells with 48/80 also significantly offset TF upregulation. In immunofluorescent approaches, FACScan analysis revealed that 48/80 had no effect on either LPS recognition or the expression of its receptors (CD14 and CD11b). Moreover, LPS-induced TF expression as well as synthesis remained unaffected in the presence of 48/80. Consistent with the independence of LPS action, 48/80 was also able to inhibit TF activity induced by A23187, ionomycin, or Quin-2 AM. Interestingly, 48/80 significantly decreased the FVII binding to either resting or LPS-challenged cells. In conclusion, our results elucidate that the inhibitory action of 48/80 was independent of LPS signaling including recognition, receptor expression, and the induced TF expression/ synthesis. However, 48/80 was able to directly block FVII binding to monocytic TF, thereby resulting in such antagonism to LPS-induced TF-initiated extrinsic coagulation.

Animals↗
PubMed1999

Blockade by polyunsaturated n-3 fatty acids of endotoxin-induced monocytic tissue factor activation is mediated by the depressed receptor expression in THP-1 cells.

BACKGROUND: Monocytic hypercoagulation often occurs in inflammatory conditions. We have previously reported that polyunsaturated n-3 fatty acids (n-3 FA) including eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6) prevent the activation of monocytic tissue factor (TF) induced by bacterial endotoxin [lipopolysaccharide (LPS)] in cell cultures and animals. HYPOTHESIS: We herein explore the mode of inhibitory action of n-3 FA to determine if LPS transmembrane signaling is blocked, exerting such antagonism. RESULTS: Exposure of human leukemia monocytic THP-1 cells to bacterial endotoxin (Escherichia coli 0111:B04, 1.5 microg/ml) for 6 h significantly activated TF activity and the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and interleukin (IL)-1beta in conditioned medium. Pretreatment with n-3 FA, 20:5 and 22:6 at 10 microM, resulted in time-dependent suppression of not only TF activation but also the elicitation of NO, TNF-alpha, and IL-1beta. These LPS responses were substantially depressed by more than 50% after a 72-h pretreatment. FACScan analysis showed that n-3 FA readily prevented fluorescein isothiocyanate (FITC)-conjugated LPS from binding to THP-1 cells by approximately 70%. The observation that anti-CD14 mAb diminished FITC-LPS binding in a dose-dependent fashion has revealed CD14 dependency in LPS recognition. LPS upregulated CD14 expression, which was significantly arrested by n-3 FA. Similarly, the upregulation of the expression of CD11b, another proposed LPS receptor, was also minimally but significantly depressed by n-3 FA. CONCLUSION: The present study demonstrates that n-3 FA are able to block LPS transmembrane signaling via suppression of the receptor upregulation, mediating a variety of significant antagonisms against LPS action.

CD11 Antigens↗
PubMed1999

Compound 48/80 suppresses monocytic tissue factor-initiated extrinsic blood coagulation induced by bacterial endotoxin.

BACKGROUND: Hypercoagulability is one of the commonly exhibited endotoxemia septic symptoms; it could contribute to the manifestation of disseminated intravascular coagulation presenting threats to cardiovascular functions. The underlying mechanism, however, remains largely complex and unknown. OBJECTIVES: We herein determine whether bacterial endotoxin (LPS) upregulates the activities of clotting factors in plasma, contributing to extrinsic hypercoagulation. Compound 48/80 (48/80) is also tested for its ability to suppress hypercoagulation. METHODS: In an in vitro infection model, we exposed whole blood to LPS (Escherichia coli 0111:B04; 100 ng/ml) for 2 h. Thrombin time (TT), prothrombin time (PT), and the activities of clotting factors ( FVII, FIX, FX ) in plasma contributing to the extrinsic coagulation were determined. Peripheral blood monocytes were isolated from Histoplaque 1077 gradient centrifugation, and the procoagulant activity was determined by a single-stage clotting assay on a Fibrometer. RESULTS: LPS drastically activated monocytic procoagulant activity which was defined as tissue factor (TF) activity, whereas LPS had no effect on TT, PT, and the activities of clotting factors in plasma. 48/80 not only instantaneously offset LPS-induced monocytic TF activation, but also significantly inhibited PT including the activities of clotting factors (FVII, FIX, and FX) in plasma, whereas TT remained unaffected. CONCLUSIONS: Monocytic TF activation was solely responsible for the extrinsic hypercoagulation in response to LPS. 48/80 effectively suppressed LPS-induced monocyticTF-initiated extrinsic coagulation at multiple sites, possibly presenting a new therapy for an instantaneous relief of hypercoagulation under septic conditions.

Blood Coagulation↗
PubMed1999

Up-regulation by human recombinant transforming growth factor beta-1 of collagen production in cultured dermal fibroblasts is mediated by the inhibition of nitric oxide signaling.

BACKGROUND: Hypertrophic scarring remains the most disabling sequela for burn survivors. Little is known about its pathogenesis. Collagen accumulation, however, has been consistently observed in burn hypertrophic scars (HS). STUDY DESIGN: We have studied collagen production in the dermal fibroblasts derived from HS, which has developed for 9 months to 2 years. Reconstructive surgery was performed to remove HS from which the fibroblasts were cultured. Similarly, the normal cells were grown from the patient's donor site (DS), which provided autografting to the HS site. Collagen production in HS and DS fibroblasts was compared and analyzed in minimal essential amino acid medium containing 5% fetal bovine serum with inclusion of L-ascorbic acid (100 microg/mL) and beta-aminopropoinitrile (100 microg/mL) by monitoring a 20-h [3H]proline incorporation into bacterial collagenase III-digestible protein in the conditioned media. RESULTS: We failed to detect any significant difference in collagen production in vitro between HS and DS. Irrespective of the fibroblasts from HS or DS, collagen production was substantially stimulated by human recombinant transforming growth factor beta-1 (TGF-beta1) (20 ng/mL) by approximately 250% after a 3-day pretreatment. In contrast, sodium nitroprusside (SNP) at 100 microM exhibited significant suppression (68%), which was rescued by hemoglobin (10 microM). TGF-beta1 significantly decreased nitric oxide (NO) production by 55%. In contrast, NO level drastically increased by 350% following SNP treatment. Epidermal growth factor showed no effect on either collagen production or NO level. The linear regression analysis shows a significant inverse correlation (r = 0.72; p < 0.05) of NO level with collagen production, suggesting the involvement of NO signaling in the modulation of collagen production. Consistent with the notion, we further showed that N-nitro-L-arginine methyl ester (100 microM) caused a synergistic stimulation and an arrested inhibition of collagen production in the presence of TGFbeta-1 and SNP, respectively. 8-BrcGMP (300 microM) mimicked the NO inhibitory action, while methylene blue (50 microM) restored the collagen production which was inhibited by SNP. Moreover, 8-BrcGMP offset the stimulation of collagen production. CONCLUSIONS: The dermal fibroblasts derived from HS were not different from normals with respect to collagen production and their responses to regulations. The inhibition of collagen production was achieved by a cGMP-dependent NO action. TGFbeta-1 inhibited NO/cGMP signaling to ensure its stimulatory effect on collagen production in the dermal fibroblasts.

Burns↗
PubMed1998

Antagonism by IL-4 and IL-10 of endotoxin-induced tissue factor activation in monocytic THP-1 cells: activating role of CD14 ligation.

BACKGROUND: Monocytic tissue factor (TF), initiating the extrinsic blood coagulation pathway, is often upregulated under septic or inflammatory conditions. The complex activating mechanism remains largely unclear and no effective strategy has been firmly established. In this study, we used a model monocytic cell line (human leukemic THP-1 promonocytes) to address (1) the nature of TF activation in response to bacterial endotoxin and (2) the application of anti-inflammatory cytokines in relieving monocytic hypercoagulation. RESULTS: TF in THP-1 cells was substantially activated by exposure to bacterial endotoxin (LPS; 5 micrograms/ml) for 6 h. Human recombinant IL-4 (500 ng/ml) and IL-10 (500 ng/ml) inhibited TF activation induced by LPS. To determine if these cytokines depressed LPS recognition resulting in such inhibition, we employed an anti-CD14 mAb (UCHM-1; Sigma Chemical) to address the role of CD14 in LPS transmembrane signaling. LPS-induced TF activation was depressed by 35% upon inclusion of the anti-CD14 mAb (1:10 dilution). This antibody alone mimicked TF activation which accounted for 35% of the LPS-induced TF activation, suggesting the activating role of CD14 ligation. In addition, the anti-CD14 mAb elicited the production of nitric oxide (NO) which was found to be independent of TF activation. NO production could serve as an independent index for monitoring LPS recognition. IL-4 depressed the anti-CD14 mAb-induced TF activation as well as NO elicitation, indicating the blockade of CD14 ligation. In contrast, IL-10 showed differential inhibitory activities. TF activation induced by either LPS or anti-CD14 mAb was inhibited by IL-10 which did not show any inhibition on NO elicitation under these conditions. In a separate approach, neither IL-4 nor IL-10 inhibited phorbol ester-induced NO elicitation. More direct evidence came from an epifluorescent demonstration showing that IL-4 blocked binding of FITC-conjugated LPS and anti-CD14 mAb to THP-1 cells. CONCLUSIONS: Taken together, the results suggest that LPS action in relation to TF activation consists of CD14-independent and -dependent signaling including CD14 ligation. We also showed that anti-inflammatory cytokines (IL-4 and -10) significantly depressed TF activation. IL-4 antagonized CD14-dependent LPS recognition leading to the depression in TF activation.

Antibodies, Monoclonal↗
PubMed1996

Burns and inhalation injury caused by steam.

Injury to the respiratory tract from inhalation of steam, although rare, often leads to acute pulmonary insufficiency. The injury to the alveolar epithelium results in increased pulmonary capillary permeability, pulmonary oedema and interfering abnormalities in gas exchange. Vigorous treatment should be instituted in these patients to obtain optimal outcome.

Acute Disease↗
PubMed1995

Perineal burns in males secondary to spouse abuse.

Male spouse abuse seems to be on the increase. Recognition of these injuries is often difficult as patients are reluctant to disclose the aetiology and press charges against the assailant. We identified a similar group of male patients who sustained burns to their external genitalia secondary to spouse abuse.

Adult↗
PubMed1995

Burns of the lymphoedematous extremity.

In most patients with lymphoedema, neither a medical nor a surgical approach can provide a cure. Due to the progressive nature of the disease, it is imperative that every attempt should be made at controlling oedema and preventing infection. As burn injury to the lymphoedematous extremity is associated with significant morbidity, we report a patient with deep burns affecting a lymphoedematous extremity.

Burns↗
PubMed1994

Metastatic adenocarcinoma in a recent burn scar.

Neoplastic lesions arising in recent burn scars are apparently quite rare and we could find no report of such an occurrence in the literature. Nevertheless we recently treated a patient with a metastatic adenocarcinoma from the lung in a 3-month-old burn scar.

Adenocarcinoma↗
PubMed1991

Prospective comparison of a bovine collagen dressing to bovine spray thrombin for control of haemorrhage of skin graft donor sites.

A prospective trial compared spray thrombin to a bovine collagen haemostatic agent on skin graft donor sites. Twenty donor sites had half of the area covered with the collagen sheet and the other half sprayed with thrombin. Each treatment was covered with a surgical sponge, pressure was applied for 10 min and then the sponges and collagen were removed. A second sponge was then placed on each wound for 2 min. These sponges were tested for oxyhaemoglobin by spectrophotometry. The results showed that the half of 18/20 donor sites covered with collagen lost more blood than the half treated with spray thrombin. These results suggest that thrombin is superior to collagen sheets for donor sites treated in this manner.

Bandages↗
PubMed1991

A review of the reconstructive surgery needs of 3167 survivors of burn injury.

A retrospective review of all patients admitted for acute care over the past 15 years was undertaken to identify readmission for reconstructive surgery. Of 3167 survivors 629 or 19.9 per cent were readmitted for at least one reconstructive procedure. Those patients requiring readmission for reconstructive surgery were younger and had larger total body surface burns compared to the group that were not readmitted for reconstructive surgery. The percentage of patients now being readmitted for reconstructive surgery is decreasing significantly and covers all size burns. The most common area of reconstruction, by frequency, involved the hand and wrist, arm and forearm, face, and neck. These data indicate that the goal of reducing the incidence of burn scar contractures in this patient population is being achieved. However, further work needs to be done to reduce the frequency of reconstructive surgery and to identify those variables which predispose patients to burn scar contractures which are amenable to intervention.

Acute Disease↗
PubMed1990

Superoxide dismutase prevents lipid peroxidation in burned patients.

Animal models and human studies have shown that conjugated dienes rise in the plasma after thermal injury. These dienes may serve as a marker of oxygen radical-mediated tissue injury. Twelve burn patients were randomized to receive the antioxidant enzyme polyethylene glycol-conjugated superoxide dismutase (PEG-SOD). Patients received either 500 or 1000 units per kilogram of PEG-SOD intravenously within 6 h of injury. Plasma samples were collected and conjugated diene levels were compared to diene levels of burn patients not treated and to diene levels from normal volunteers. Conjugated diene levels were increased in burn patients. PEG-SOD in either dose initially decreased conjugated diene levels in the plasma of both treatment groups. By 72 h, the diene levels increased in the 500 unit/kg group, but remained at near control levels in the 1000 unit/kg group for up to 200 h after injury. These data suggest that PEG-SOD is capable of preventing conjugated dienes formed as the result of oxygen radical production. It appears that 1000 units/kg is more effective than 500 units/kg in preventing conjugated diene formation.

Adult↗
PubMed1990

Rehabilitation of burned patients with bilateral above-knee amputations.

Bilateral above the knee amputations occur rarely in the seriously burned population. In a 6-month interval, two patients were treated in our Burn Center whose trauma necessitated bilateral above-knee amputations. This led us to review our experience with such cases. A review of 5018 patients treated over a 28-year period identified six patients over a 20-year period who had bilateral above-knee amputations, four of whom survived. These patients were found to have large and deep burns, concomitant injuries and a number of complications. Recovery was time consuming and costly. Physical therapists were challenged to create adaptations of traditional methods to meet the rehabilitation needs of these patients. The patients who survived remain wheelchair bound and unemployed, but are socially active, appear relatively well adjusted and report themselves satisfied with their lives. During a period of time when technological advances in medicine are ensuring survival of victims of massive trauma, questions are raised concerning the advisability of resuscitation. Knowledge gained in the treatment of these patients who are both burned and have had bilateral above-knee amputations can be reassuring to care providers with limited experience with such massive injury.

Adult↗
PubMed1990

Metabolic alterations in burn patients: detection of adenosine triphosphate degradation products and lipid peroxides.

Seven patients admitted to the University of Michigan Burn Center with greater than 20 per cent total body surface area burns were studied for evidence of oxygen radical production, as demonstrated by serum lipid peroxides, and adenosine triphosphate (ATP) degradation, based upon ATP degradation products in blood (serum purines) and urine (urine purines and urine uric acid). Lipid peroxides (conjugated dienes) were elevated beginning on day 1 postburn and remained elevated up to day 5, the duration of the study. ATP degradation products were elevated during the initial 24 h postburn but rapidly fell to normal levels. This study provides evidence of biochemical alterations in thermally injured patients similar to observations in animal models which demonstrate that ATP degradation and the production of oxygen radicals are part of the initial response to thermal trauma.

Adenosine Triphosphate↗
PubMed1990

Topical antimicrobial testing of burn wound microorganisms: evaluation of a new technique.

A new system of determining susceptibility of burn wound microorganisms to topical antimicrobial therapy was studied. The new method, TOPITEST, employs a methacrylate vehicle impregnated with various antimicrobial agents and an agar overlay. Test plates can be inoculated by a direct swab culture of the wound or with micro-organisms isolated by other methods. This new technique was compared to an agar well diffusion (AWD) method using commercially compounded antimicrobials and biopsy-recovered organisms. The new test method provided comparable results to the more established AWD test when using biopsy-recovered organisms. Using the swab culture technique, however, TOPITEST had a high rate of failure to recover sufficient organisms from the burn wound for testing. The new, more standardized test provided results earlier than the AWD test, but may prove to be less cost effective.

Administration, Topical↗