| 2018 | A Case of Generalized, Superinfected Dermatitis and Inguinal Mycobacterium Lymphadenitis - TB or not TB? | Acta Dermatovenerol Croat | Dear Editor, Eczema is an inflammatory dermatitis mediated by cellular immunity, with an etiology in which environmental, immunological, and genetic factors are involved. Skin inflammation through proinflammatory cytokines creates a favorable environment for microbial antigens and optimal conditions for infection (1). In case of underlying immunosuppression, inflammatory features of dermatitis and superimposed infections are more severe. The presence of minor trauma of the skin in the form of fissures can favor both easier inoculation of some bacterial germs, leading to a dermatitis superinfection, and/or the transcutaneous inoculation of atypical mycobacteria, with a possibility of developing localized types of tuberculous lymphadenitis (TLA). TLA, the localized type of systemic tuberculosis (TB) infection, is the most common form of extra-pulmonary TB in developing countries (2), while lymphadenitis due to atypical mycobacteria is a localized disease, more frequently seen in developed countries (3,4). In tuberculosis, the transmission of Mycobacterium tuberculosis is airborne, while in atypical mycobacterium lymphadenitis transmission can be both airborne or by ingestion or inoculation (5). In both forms of TB, lymphadenopathy evolves towards abscess and presents fibrotic scars or calcifications upon healing (6). A positive diagnosis involves a clinical and epidemiological investigation, a purified protein derivative (PPD) skin test, ultrasound, and CT / MRI of lymph node masses. A lymph node biopsy is used to confirm the diagnosis of TB and PCR, while positive culture confirms the etiology of TB lymphadenitis. The differential diagnosis of TLA is difficult: neoplastic, bacterial, or viral and fungal infections, sarcoidosis, Castleman's disease, drug reactions, etc. (5). TB-induced immunosuppression may favor the development of fungal and bacterial infections, sometimes severe and poorly responsive to treatment. On the other hand, immunosuppressive conditions increase the risk of extra-pulmonary TB (2). A 40-year old woman who had experienced recurrent episodes of dermatitis over the previous 7 years was hospitalized with fever, malaise, and a disseminated erythematous and crusted, exudative, and flexural itching rash (Figure 1). There were fetid, purulent secretions at the conjunctival, auricular, genital, and umbilical areas. The clinical exam also revealed lymphadenopathy syndrome (large, painful submandibular, cervical, and axillar bilateral lymph nodes; an indurated, painful, and adherent left inguinal lymph node of 5-6 cm). Microbial cultures isolated multiple multi-drug-resistant bacteria (SAH-MRSA, Acinetobacter baumannii, Enterococcus faecalis, E. coli, Enterobacter) and Candida albicans in the oral cavity and conjunctival, auricular, nasal, umbilical, and genital areas. The skin biopsy confirmed the diagnosis of dermatitis. PPD skin test was 21 mm. Other tests (HIV and syphilis serology, blood culture, chest X-ray) were negative. Systemic treatment with vancomycin, metronidazole, fluconazole, local antiseptic compresses, and topical corticosteroid ointments was initiated. 2 days after starting the treatment with vancomycin, Redman syndrome occurred (headache, dyspnea, colicky pains, myalgia, rush, fever (39 °C), hypotension (80/40 mmHg), and tachycardia (100 bpm)). This syndrome resolved upon discontinuation of Vancomycin. Further treatment with imipenem/cilastatinand linezolid for 14 days lead to a favorable response with amelioration of the symptoms. Biopsy of the submandibular lymph node raised the suspicion of Castleman's disease; however, due to the overall incomplete clinical picture (no night sweats, no weight reduction, lack of hepatosplenomegaly and peripheral neuropathy), we decided to perform a biopsy of an inguinal lymph node. The histopathological aspect suggested TLA (lymphoid hyperplasia predominantly diffuse, reactive, presenting tuberculous follicles with central caseous necrosis) (Figure 2). A combination of specific antituberculous drugs (isoniazid, rifampicin, pyrazinamide, and ethambutol) for 6 months resolved the lymphadenopathy syndrome with no further recurrence of eczema and skin infections. Certain delayed hypersensitivity mechanisms are involved both in dermatitis and in TB. CD4 lymphocytes are the primary mediators of anti-TB immunity, while proinflammatory cytokines mediate the activation of macrophages involved in controlling bacillary growth (1). In cases of superinfected dermatitis, microbial exotoxins penetrate the skin barrier more easily due to inflammation. Released cytokines (IL-1, TNF, and IL12) favor the expression of E-selectin on endothelial vascular growth factor and on skin lymphocyte antigen expression, with amplification of initial skin inflammation and creating favorable conditions for microbial colonization and infection (7). The common denominator in dermatitis and TB are the circulating immune complexes (up to 56% of TB cases), which are formed by the interaction between an antibody and bacterial antigen (8), which was in this case evidenced by increased levels of IgA and IgG. In our case, the frequent recurrences of infected dermatitis with multiple multi-drug-resistant germs that were poorly responsive to treatment and displayed a severe evolution towards generalization as well as the lymphadenopathy and the persistence of a biological inflammatory syndrome indicated that another immunosuppressive cause could be involved. Isolated bacterial and fungal germs changed the immune status of the patient. The risk of mycobacterium infection was increased by the environment they created and the patient's underlying skin inflammation. The diagnosis of TB lymphadenitis was established by the histopathologist, but in the absence of PCR we could not determine whether the TB infection was caused by Mycobacterium tuberculosis or by atypical mycobacteria. Given that there was no evidence of other sites of TB infection, we conjectured that inoculation of mycobacterium took place at the skin lesion and that an atypical mycobacterium might have contributed to the etiology of the TLA. In our case, the anti-tuberculous drugs and skin infection treatment with follow-up of the side-effects led to complete remission of mycobacterium lymphadenitis, dermatitis, and infectious processes, without relapses. In conclusion, in the present case chronic dermatitis alongside infection with multi-drug-resistant germs led to an immunosuppressive status which, when associated with the presence of multiple skin ports of entry, allowed a mycobacterial infection at the inguinal lymph node level. Inguinal TLA induced severe dermatitis and difficulties in diagnosis and treatment. |
| 2015 | Attenuating systemic inflammatory markers in simulated high-altitude exposure by heat shock protein 70-mediated hypobaric hypoxia preconditioning in rats. | J Formos Med Assoc | The primary goal of this study was to test whether high-altitude exposure (HAE: 0.9% O(2) at 0.47 ATA for 24 hours) was capable of increasing the systemic inflammatory markers as well as the toxic organ injury indicators in rats, with a secondary goal to test whether preinduction of heat shock protein (HSP) 70 by hypobaric hypoxia preconditioning (HHP: 18.3% O(2) at 0.66 ATA for 5 h/day on 5 days consecutively for 2 weeks) attenuated the proposed increased serum levels of both the systemic inflammatory markers and the toxic organ injury indicators.Rats were assigned to: (1) non-HHP (21% O(2) at 1.0 ATA)+non-HAE (21% O(2) at 1.0 ATA) group; (2) non-HHP+HAE group; (3) HHP+non-HAE group; (4) HHP+HAE group; and (5) HHP+HSP70 antibodies (Ab)+HAE group. For the HSP70Ab group, a neutralizing HSP70Ab was injected intravenously at 24 hours prior to HAE. All the physiological and biochemical parameters were obtained at the end of HAE or the equivalent time period of non-HAE. Blood samples were obtained for determination of both the systemic inflammatory markers (e.g., serum tumor necrosis factor-α, interleukin-1β, E-selectin, intercellular adhesion molecule-1, and liver myeloperoxidase activity) and the toxic organ injury indicators (e.g., nitric oxide metabolites, 2,3-dihydroxybenzoic acid, and lactate dehydrogenase).HHP, in addition to inducing overexpression of tissue HSP70, significantly attenuated the HAE-induced hypotension, bradycardia, hypoxia, acidosis, and increased tissue levels of both the systemic inflammatory markers and the toxic organ injury indicators. The beneficial effects of HHP in inducing tissue overexpression of HSP70 as well as in preventing the HAE-induced increased levels of the systemic inflammatory markers and the toxic organ injury indicators could be significantly reduced by HSP70Ab preconditioning.These results suggest that HHP may downgrade both the systemic inflammatory markers and the toxic organ injury indicators in HAE by upregulating tissue HSP70. |
| 2013 | Oral propranolol for retinopathy of prematurity: risks, safety concerns, and perspectives. | J Pediatr | To evaluate safety and efficacy of oral propranolol administration in preterm newborns affected by an early phase of retinopathy of prematurity (ROP).Fifty-two preterm newborns with Stage 2 ROP were randomized to receive oral propranolol (0.25 or 0.5 mg/kg/6 hours) added to standard treatment or standard treatment alone. To evaluate safety of the treatment, hemodynamic and respiratory variables were continuously monitored, and blood samples were collected weekly to check for renal, liver, and metabolic balance. To evaluate efficacy of the treatment, the progression of the disease (number of laser treatments, number of bevacizumab treatments, and incidence of retinal detachment) was evaluated by serial ophthalmologic examinations, and plasma soluble E-selectin levels were measured weekly.Newborns treated with propranolol showed less progression to Stage 3 (risk ratio 0.52; 95% CI 0.47-0.58, relative reduction of risk 48%) or Stage 3 plus (relative risk 0.42 95% CI 0.31-0.58, relative reduction of risk 58%). The infants required fewer laser treatments and less need for rescue treatment with intravitreal bevacizumab (relative risk 0.48; 95% CI 0.29-0.79, relative reduction of risk 52 %), a 100% relative reduction of risk for progression to Stage 4. They also had significantly lower plasma soluble E-selectin levels. However, 5 of the 26 newborns treated with propranolol had serious adverse effects (hypotension, bradycardia), in conjunction with episodes of sepsis, anesthesia induction, or tracheal stimulation.This pilot study suggests that the administration of oral propranolol is effective in counteracting the progression of ROP but that safety is a concern. |
| 2012 | Indoleamine-2,3-dioxygenase activity in experimental human endotoxemia. | Exp Transl Stroke Med | Excessive tryptophan metabolism to kynurenine by the rate-limiting enzyme endothelial indoleamine 2,3-dioxygenase 1 (IDO) controls arterial vessel relaxation and causes hypotension in murine endotoxemia. However, its relevance in human endotoxemia has not been investigated so far. We thus aimed to study changes in blood pressure in parallel with tryptophan and kynurenine levels during experimental endotoxemia in humans.Six healthy male volunteers were given E. coli lipopolysaccharide (LPS; 4 ng/kg) as a 1-min intravenous infusion. They had levels of soluble E-Selectin and soluble vascular cell adhesion molecule-1 as well as IDO activity assessed as the kynurenine-to-tryptophan plasma ratio by liquid chromatography-tandem mass spectrometry at various time points during a 24 h time course. During endotoxemia, IDO activity significantly increased, reaching peak levels at 8 h after LPS infusion (44.0 ± 15.2 vs. 29.4 ± 6.8 at baseline, P<0.0001). IDO activity correlated inversely with the development of hypotension as shown by random effects linear regression models. Finally, IDO activity exhibited a kinetic profile similar to that of soluble endothelial-specific adhesion molecules.LPS is a triggering factor for the induction of IDO in men. Our findings strongly support the concept that the induction of IDO in the vascular endothelium contributes to hypotension in human sepsis. |
| 2011 | Endothelial cell activation in emergency department patients with sepsis-related and non-sepsis-related hypotension. | Shock | Previous studies found increased circulating levels of biomarkers related to endothelial cell activation in patients with sepsis, particularly in the most severe sepsis stages of sepsis shock. It remains unclear, however, whether this activation is mainly driven by sepsis-specific mechanisms or occurs as a generalized inflammatory response. The objective of this analysis was to compare patterns of biomarkers of endothelial cell activation in patients with hypotension due to sepsis and nonsepsis etiologies. This is a secondary analysis of a prospective, observational cohort study including emergency department patients older than17 years with an episode of hypotension defined as any systolic blood pressure measurement less than 100 mmHg. Etiology of hypotension episodes was classified as sepsis or nonsepsis (eg, cardiac or hemorrhagic). Endothelial activation biomarkers of cell adhesion (E-selectin, vascular cell adhesion molecule 1 [VCAM-1], and intercellular adhesion molecule 1 [ICAM-1]), coagulation (plasminogen activator inhibitor 1 [PAI-1]), and vascular endothelial growth factor (VEGF) signaling (VEGF, soluble fms-like tyrosine kinase 1 [sFLT-1]) were assayed. A total of 161 patients were analyzed. Hypotension was classified as sepsis (n = 69), nonsepsis (cardiac [n = 35], hemorrhagic [n = 12]), or indeterminate (n = 45). With the exception of PAI-1, median plasma levels of all endothelial markers were significantly higher in patients with sepsis compared with nonsepsis etiology (P < 0.05 for all comparisons). Logistic regression analysis, adjusted for age, sex, mean blood pressure level, and mortality, confirmed a significant association of E-selectin (odds ratio [OR], 3.7; 95% confidence interval [CI], 1.7-7.8, P < 0.001) and sFLT-1 (OR, 2.0; CI, 1.1-3.8; P < 0.03) with sepsis etiology. Biomarkers VCAM-1 (OR, 2.0; CI, 0.88-4.4; P = 0.1), VEGF (OR, 1.5; CI, 0.98-2.2; P = 0.06), ICAM-1 (OR, 1.5; CI, 0.9-2.6; P = 0.2), and PAI-1 (OR, 1.4; CI, 0.8-2.3; P = 0.2) did not reach statistical significance. This study found a sepsis-specific activation of endothelium activation markers, particularly E-selectin and sFLT-1, in emergency department patients with hypotension. |
| 2011 | Therapeutic effects of melatonin on heatstroke-induced multiple organ dysfunction syndrome in rats. | J Pineal Res | Melatonin reportedly exerts beneficial effects to attenuate multiple organ dysfunction syndrome (MODS) in septic shock. Heatstroke resembles septic shock in many aspects. Thus, this study was performed on the anesthetized rats by using heat exposure to induce heatstroke-associated MODS. We evaluated the effect of melatonin, a versatile molecule synthesized in the pineal gland and in many organs, in heatstroke rats and showed that melatonin (0.2-5.0 mg/kg of body weight, i.v., immediately after the start of heat stress) significantly (i) attenuated hyperthermia, hypotension and hypothalamic ischemia and hypoxia, (ii) reduced plasma index of the toxic oxidizing radicals like nitric oxide metabolites and hydroxyl radicals, (iii) diminished plasma index of hepatic and renal dysfunction like creatinine, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase, (iv) attenuated plasma systemic inflammation response molecules like soluble intercellular and lesion molecule-1, E-selectin, tumor necrosis factor-alpha, interleukin (IL)-1β, and IL-6, (v) promoted plasma levels of an anti-inflammatory cytokine IL-10, (vi) reduced an index of infiltration of polymorphonuclear neutrophils in the lung like myeloperoxidase activity, and (vii) promoted the survival time to fourfold compared with the heatstroke alone group. Thus, melatonin could be a novel agent for the treatment of heatstroke animals or patients in the early stage. |
| 2010 | A prospective multicenter cohort study of the association between global tissue hypoxia and coagulation abnormalities during early sepsis resuscitation. | Crit Care Med | Coagulation activation is an integral part of sepsis pathogenesis. Experimental data suggest that endothelial exposure to hypoxia activates coagulation. We aimed to test the hypothesis that the quantity of exposure to global tissue hypoxia is associated with the degree of coagulation activation during early sepsis resuscitation.Prospective, multicenter cohort study.Emergency department and intensive care unit of three academic hospitals.Inclusion criteria were age older than 17, acute infection with two or more signs of systemic inflammation, hypotension despite fluid challenge (or lactate >4 mM), and continuous central venous oxygen saturation (Scvo2) monitoring for quantitative resuscitation. Exclusion criteria were anticoagulant or blood product administration.We recorded central venous oxygen saturation continuously for 0 to 6 hrs of resuscitation and calculated the area under the curve for central venous oxygen saturation <70%. We defined hypoxia exposure as exceeding the median area under the curve for the entire cohort. At 0, 6, and 24 hrs, we measured conventional coagulation biomarkers plus thrombin-antithrombin complex, plasmin-antiplasmin complex, tissue plasminogen activator, plasminogen activator inhibitor-1, protein C, antithrombin, and endothelial markers (E-selectin, intracellular adhesion molecule-1, thrombomodulin). We compared changes during 0 to 6 hrs and 0 to 24 hrs in biomarkers between hypoxia exposure and nonexposure groups. We enrolled 40 patients (60% requiring vasopressors; 30% mortality). We found that exposure to hypoxia alone was not associated with a significant degree of coagulation activation. However, in secondary analyses we found that exposure to arterial hypotension induced E-selectin and thrombin-antithrombin complex, whereas concomitant exposure to both hypotension and hypoxia was associated with amplification of E-selectin and thrombomodulin, and a reduction in protein C.In this sample of patients undergoing quantitative resuscitation for sepsis, we found that exposure to global tissue hypoxia (as quantified by low central venous oxygen saturation) was not associated with major coagulation activation. Further investigation to elucidate the clinical factors that trigger or intensify the procoagulant response to sepsis is warranted. |
| 2009 | Human umbilical cord blood cells protect against hypothalamic apoptosis and systemic inflammation response during heatstroke in rats. | Pediatr Neonatol | Intravenous administration of human umbilical cord blood cells (HUCBC) has been shown to improve heatstroke by reducing arterial hypotension as well as cerebral ischemia and damage in a rat model. To extend these findings, we assessed both hypothalamic neuronal apoptosis and systemic inflammatory responses in the presence of HUCBCs or vehicle medium immediately after initiation of heatstroke.Anesthetized rats, immediately after the initiation of heat stress, were divided into two groups and given either serum-free lymphocyte medium (0.3mL per rat, intravenously) or HUCBCs (5 x 10(6) in 0.3 mL serum-free lymphocyte medium, intravenously). Another group of rats were exposed to room temperature (26 degrees C) and used as normothermic controls. Heatstroke was induced by exposing the anesthetized rats to a high ambient temperature of 43 degrees C for 68 minutes.After the onset of heatstroke, animals treated with serum-free lymphocyte medium displayed hyperthermia, hypotension, bradycardia, hypothalamic neuronal apoptosis and degeneration, and up-regulation of systemic inflammatory response molecules including serum tumor necrosis factor-alpha, soluble intercellular adhesion molecule-1 and E-selectin. Heatstroke-induced hypotension, bradycardia, hypothalamic neuronal apoptosis and degeneration, and increased systemic inflammatory response molecules were significantly inhibited by HUCBC treatment. Although heatstroke-induced hyperthermia was not affected by HUCBC treatment, the serum levels of the anti-inflammatory cytokine interleukin-10 were significantly increased by HUCBC therapy during hyperthermia.These findings suggest that HUCBC transplantation may prevent the occurrence of heatstroke by reducing hypothalamic neuronal damage and the systemic inflammatory responses. |
| 2000 | Systemic inflammation in hemorrhagic fever with renal syndrome correlates with hypotension and thrombocytopenia but not with renal injury. | J Infect Dis | Systemic inflammation is common in patients with nephropathia epidemica (NE), a European form of hemorrhagic fever. Markers of inflammation were studied in a patient with NE with respiratory insufficiency (patient 1), 18 other patients with NE, and 13 patients with a viral infectious disease other than NE. Neutrophil and monocyte CD11b expression levels, determined by flow cytometry; soluble interleukin (IL)-2 receptor (sIL-2R), IL-6, and IL-8 concentrations, determined by means of Immulite; and soluble E-selectin, determined by ELISA, were higher in patients with NE than in healthy subjects. The findings were not specific for NE and did not correlate with serum creatinine levels, but the findings correlated inversely with mean arterial pressure (sIL-2R and monocyte CD11b expression) and minimum platelet count (sIL-2R, IL-6, neutrophil, and monocyte CD11b expression). Monocyte CD11b expression in patient 1 was extremely high, suggesting that monocytes may contribute to development of lung injury. Severity of inflammation in patients with NE is related to hypotension and platelet consumption but not to renal injury. |
| 1998 | Effects of explosive brain death on cytokine activation of peripheral organs in the rat. | Transplantation | The success rate of transplanted organs from brain-dead cadaver donors is consistently inferior to that of living sources. As cadaver and living unrelated donors are equally genetically disparate with a given recipient, the difference must lie within the donor himself and/or the effects of organ preservation and storage. We have hypothesized that irreversible central nervous system injury may up-regulate proinflammatory mediators and cell surface molecules in peripheral organs to be engrafted, making them more prone to host inflammatory and immunological responses.Rats undergoing surgically induced acutely increased intracranial pressure (explosive brain death) were followed for 6 hr. Their peripheral tissues were examined by reverse transcriptase polymerase chain reaction and immunohistology, serum factors were assessed by enzyme-linked immunosorbent assay, and the influence of inflammatory molecules in the blood stream was determined by cross-circulation experiments with normal animals.mRNA expression of both lymphocyte- and macrophage-associated products increased dramatically in all tissues. Similar factors in serum were coincidentally increased; these were shown to be active in vivo by cross-circulation with normal animals. The organs of all control groups, including animals with important ischemic injury and with hemorrhagic shock, were negative. Up-regulation of MHC class I and II antigens and the co-stimulatory molecule B7 suggests increased immunogenicity of the peripheral organs. These changes could be inhibited by: (i) administration of a recombinant soluble P-selectin glycoprotein ligand-Ig, a P- and E-selectin antagonist; and (ii) a fusion protein, cytotoxic T lymphocyte antigen 4-Ig, which blocks B7-mediated T-cell co-stimulation.Activation of peripheral organs following explosive brain death may be caused by various interrelated events, including the effects of massive acute central injury, hypotension, and circulating factors. Almost complete suppression of these changes could be produced by biological agents. Such interventions, if reproducible in humans, could improve the quality of organs from "marginal" donors, broadening the criteria for donor acceptance. |
| 1996 | Increased concentrations of cytokines and adhesion molecules in patients after repair of abdominal aortic aneurysm. | Eur J Surg | To evaluate the association between inflammatory mediators and clinical outcome in patients after repair of abdominal aortic aneurysms.Prospective study.University hospital, The Netherlands.30 Consecutive patients who had undergone elective or acute repair of abdominal aortic aneurysms.Plasma concentrations of the cytokines tumour necrosis factor (TNF), interleukin-6 (IL-6) and interleukin-8 (IL-8) as well as soluble TNF receptors and the soluble (s) adhesion molecules E-selectin and intercellular adhesion molecule 1 (ICAM-1) were measured and correlated with the degree of systemic hypotension (shock: hypotension more than 15 minutes) and clinical outcome.Peak plasma concentrations of TNF and IL-6 were significantly higher in shocked patients (p < 0.005 and p < 0.0005, respectively) and those who died (both p < 0.01), whereas concentrations of IL-8 increased only when shock complicated rupture of the aneurysm (p < 0.01). Increases in the concentrations of TNF receptors reflected impaired renal function. In contrast to sE-selectin concentrations, peak sICAM-1 concentrations were significantly higher in shocked patients (p < 0.01) and those that died (p < 0.01).These results strongly suggest that increased concentrations of sICAM-1 and IL-6 reflect the inflammatory response induced by ischaemia after repair of an abdominal aortic aneurysm, and indicate that the postoperative course is likely to be complicated. |
| 1993 | Soluble E-selectin is found in supernatants of activated endothelial cells and is elevated in the serum of patients with septic shock. | J Immunol | A quantitative sandwich ELISA for E-selectin in the fluid phase (soluble E-selectin, sEs) has been developed that is sensitive to 100 pg/ml. The assay shows no reactivity with either L- or P-selectins. We have used this to determine the fate of E-selectin after cell-surface expression and to test whether levels measured in vivo may represent the state of endothelial activation. E-selectin was first detectable in supernatants of IL-1-stimulated endothelial cells at 24 h, and increased slowly up until 72 h. However, over this time period the total E-selectin detectable in the system (cells plus supernatants) declined dramatically. 125I-surface-labeled endothelial cells cultured for 24 h show an E-selectin of reduced m.w. in the supernatant, indicating that the molecule is shed from the surface. The shed form also appears to be slightly smaller than the intact membrane form as determined from immunoprecipitation and molecular sieving studies. In addition, the cytoplasmic domain of the molecule found in supernatants of activated endothelial cells and in serum is not intact as determined by loss of reactivity with an antipeptide antibody specific for the cytoplasmic domain. We have examined the sera of 71 normal individuals. Without exception, sEs was found in serum in the range of 0.13 to 2.8 ng/ml, suggesting that even in the absence of overt inflammatory processes E-selectin is being synthesized and released into the bloodstream. In addition, bacteremic patients with hypotension, but not those without, showed markedly elevated sEs values. As determined by cell-binding studies, the blood-derived form of E-selectin is biologically active. |