Screening Serum Biomarkers for Rats Preconditioned with Hyperbaric Oxygen: Potential of Predicting Prognosis for Stroke.

BACKGROUND: Stroke is a major health concern and a leading cause of mortality and morbidity. We and other groups have documented that hyperbaric oxygen preconditioning could significantly alleviate neuronal damage in ischemia‒reperfusion models through various mechanisms. However, we found that some of the subjects did not benefit from preconditioning with hyperbaric oxygen. The preconditioning phenomenon is similar to vaccination, in which the endogenous survival system is activated to fight against further injuries. However, with vaccine inoculations, we could test for specific antibodies against the pathogens to determine if the vaccination was successful. Likewise, this experiment was carried out to explore a biomarker that can reveal the effectiveness of the preconditioning before neuronal injury occurs. METHODS: Middle cerebral artery occlusion (MCAO) was used to induce focal cerebral ischemia-reperfusion injury. 2D-DIGE-MALDI-TOF-MS/MS proteomic technique was employed to screen the differentially expressed proteins in the serum of rats among the control (Con) group (MCAO model without hyperbaric oxygen (HBO) preconditioning), hyperbaric oxygen protective (HBOP) group (in which the infarct volume decreased after HBO preconditioning vs. Con), and hyperbaric oxygen nonprotective (HBOU) group (in which the infarct volume remained the same or even larger after HBO preconditioning vs. Con). Candidate biomarkers were confirmed by western blot and enzyme linked immunosorbent assay (ELISA), and the relationship between the biomarkers and the prognosis of cerebral injury was further validated. RESULTS: Among the 15 differentially expressed protein spots detected in the HBOP group by Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), 3 spots corresponding to 3 different proteins (haptoglobin, serum albumin, and haemopexin) products were identified by MALDI-TOF-MS/MS. Serum albumin and haemopexin were upregulated, and haptoglobin was downregulated in the HBOP group (p < 0.05 vs. Con and HBOU groups). After the western blot study, only the changes in haemopexin were validated and exhibited similar changes in subjects from the HBOP group in accordance with MALDI-TOF-MS/MS proteomic analysis and enzyme linked immunosorbent assay (ELISA) analysis. The serum level of the hemopexin (HPX) at 2 h after HBO preconditioning was correlated with the infarct volume ratio after MCAO. CONCLUSIONS: Haemopexin may be developed as a predictive biomarker that indicated the effectiveness of a preconditioning strategy against cerebral ischaemic injury.

Hemolysis, free hemoglobin toxicity, and scavenger protein therapeutics.

During hemolysis, erythrophagocytes dispose damaged red blood cells. This prevents the extracellular release of hemoglobin, detoxifies heme, and recycles iron in a linked metabolic pathway. Complementary to this process, haptoglobin and hemopexin scavenge and shuttle the red blood cell toxins hemoglobin and heme to cellular clearance. Pathological hemolysis outpaces macrophage capacity and scavenger synthesis across a diversity of diseases. This imbalance leads to hemoglobin-driven disease progression. To meet a void in treatment options, scavenger protein-based therapeutics are in clinical development.

Omega-3 Fatty Acids Responsive Proteins and Reduction in Breast Density in Obese Postmenopausal Women.

We reported that breast density (BD) was inversely correlated with the plasma level of DHA in postmenopausal obese, but not in nonobese, women given Lovaza (n-3FA). To identify protein biomarkers for the possible differential effect of n-3FA on BD between obese and nonobese women, an iTRAQ method was performed to analyze plasma from obese and lean women at each time point (baseline, 12 and 24-months, n = 10 per group); 173 proteins with >95% confidence (Unuses Score >1.3 and local false discovery rate estimation <5%) were identified. Comparative analysis between various groups identified several differentially expressed proteins (hemopexin precursor, vitamin D binding protein isoform 1 precursor [VDBP], fibronectin isoform 10 precursor [FN], and α-2 macroglobulin precursor [A2M]). Western blot analysis was performed to verify the differential expression of proteins in the iTRAQ study, and those found to be altered in a tumor protective fashion by an n-3FA rich diet in our previous preclinical study; gelsolin, VDBP, and FN were altered by n-3FA in a manner consistent with reduction in inflammation in obese women. To test the impact of our findings on breast cancer risk reduction by n-3FA, a posthoc analysis revealed that n-3FA administration reduced BD selectively in obese postmenopausal women.

When might transferrin, hemopexin or haptoglobin administration be of benefit following the transfusion of red blood cells?

PURPOSE OF REVIEW: After transfusion, a percentage of red blood cells undergo hemolysis within macrophages. Intravascular exposures to hemin and hemoglobin (Hb) can occur after storage bag hemolysis, some transfusion reactions, during use of medical assist devices and in response to bacterial hemolysins. Proteins that regulate iron, hemin and Hb either become saturated after iron excess (transferrin, Tf) or depleted after hemin (hemopexin, Hpx) and Hb (haptoglobin, Hp) excess. Protein saturation or stoichiometric imbalance created by transfusion increases exposure to non-Tf bound iron, hemin and Hb. Tf, Hpx and Hp are being developed for hematological disorders where iron, hemin and Hb contribute to pathophysiology. However, complexed to their ligands, each represents a potential iron source for pathogens, which may complicate the use of these proteins. RECENT FINDINGS: Erythrophagocytosis by macrophages and processes of cell death that lead to reactive iron exposure are increasingly described. In addition, the effects of transfusion introduced circulatory hemin and Hb are described in the literature, particularly following large volume transfusion, infection and during concomitant medical device use. SUMMARY: Supplementation with Tf, Hpx and Hp suggests therapeutic potential in conditions of extravascular/intravascular hemolysis. However, their administration following transfusion may require careful assessment of concomitant disease.

Is cupping blister harmful?-A proteomical analysis of blister fluid induced by cupping therapy and scald.

OBJECTIVE: Cupping therapy has a long history in traditional medicine especially in Asian countries. It was controversial whether cupping induced blisters are beneficial to healing effects, and the formation and content in the blisters remain unexplored. We aimed to identify and compare the molecular components of the blister fluid from the cupping therapy and the scalds to explore the necessary of inducing cupping induced blisters. METHODS: Fluid sample of blisters from fifteen patients receiving cupping therapy (Cupping group) and scald burns (Scald group) were collected in this study. Proteins from the blisters were separated by two-dimensional electrophoresis (2D-gel) and further analyzed by mass spectrometry. In addition, the changes in particular proteins were confirmed by Western blotting. RESULTS: The protein components are significantly different between blister from cupping therapy and scalds. The immune responses, oxidative stress and metabolic related proteins (Ig lambda-2 chain C regions, Ig gamma-1 chain C region, hemopexin, prdx2, calmodulin, succinyl-CoA ligase and tetranectin) were increased, whereas the hemoglobin subunit beta was decreased in the Cupping group compared with the Scald group. CONCLUSIONS: Cupping induced blisters contain several proteins which relate to the activation of certain immune pathways including anti-oxidation, anti-apoptosis, tissue repairing and metabolic regulation. This proteomic analysis may indicate a significant clue to the mechanism study of cupping.

The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus).

Wap65 is a molecule similar to the mammalian hemopexin that is a serum glycoprotein produced mainly by the liver with high affinity to heme. Its primary role is participating in iron metabolism scavenging heme that is released into the plasma and transporting it to the liver. It has been reported an important role of hemopexin in the inflammation as an acute-phase protein and its production is up-regulated by pro-inflammatory cytokines. There are also some evidences suggesting this immune-induction in fish Wap65 genes. Most teleost species presents two Wap65 genes but their physiological functions have not been completely elucidated; in fact, the transcriptional patterns of Wap65 genes to stimulatory treatments are variable and contradictory. In the present study two Wap65 genes, Wap65-1 and Wap65-2, have been characterized for the first time in turbot (Scophthalmus maximus). Their constitutive expression and differential modulation by thermal treatments, immune challenges (bacterial and viral), as well as iron supplementation, have been investigated. Both genes were mainly expressed in liver, but they were detected in all tested tissues. Whereas Wap65-1 and Wap65-2 were up-regulated by temperature rise and bacterial challenge, VHSV infection inhibited the expression of both genes. Moreover, iron-dextran administration induced only the overexpression of Wap65-1. Interestingly, these induction were observed in head kidney buy not in liver. The effect of Wap65 protein purified from turbot serum by hemin-agarose affinity chromatography was also studied to demonstrate a possible anti-inflammatory role, analyzing its inhibitory effect on leucocytes migration induced by zymosan injection to the peritoneal cavity.

Modulation of warm-temperature-acclimation-associated 65-kDa protein genes (Wap65-1 and Wap65-2) in mud loach (Misgurnus mizolepis, Cypriniformes) liver in response to different stimulatory treatments.

Two paralogous isoform cDNAs of warm-temperature-acclimation-associated 65-kDa protein (Wap65-1 and Wap65-2) were isolated from the cypriniform species, mud loach (Misgurnus mizolepis), and characterized. The deduced amino acid sequences of the two mud loach Wap65 isoforms (mlWap65-1 and mlWap65-2) share moderate levels of sequence homology with their corresponding orthologues from teleosts and with human hemopexin, a possible mammalian homologue. Both isoforms display conserved features, including essential motifs and/or residues that are important for the protein structure of hemopexin. In overall, mlWap65-2 is more homologous to human hemopexin than is mlWap65-1. Both mud loach Wap65 transcripts are predominantly expressed in liver, although the transcripts are ubiquitously detectable in most tissues with variable basal expression. Both mlWap65 isoforms are differentially regulated during embryonic development, and the changes in transcript levels during embryogenesis are greater for mlWap65-2 than for mlWap65-1. The transcription of the mlWap65 genes is differentially modulated by various stimuli, including thermal changes, immune challenge (lipopolysaccharide injection or bacterial infection), and heavy metal exposure (cadmium, copper, or nickel). The isoform mlWap65-1 is more responsive to warm temperature treatments than mlWap65-2, whereas mlWap65-2 is much more strongly stimulated by immune and heavy metal challenges than is mlWap65-1. Taken together, the results of this study suggest that mud loach Wap65 isoforms are potentially involved in multiple cellular pathways and that the two mud loach Wap65 isoforms undergo functional partitioning or subfunctionalization.

Dietary oil modifies the plasma proteome during aging in the rat.

Fatty acids and other components of the diet may modulate, among others, mechanisms involved in homeostasis, aging, and age-related diseases. Using a proteomic approach, we have studied how dietary oil affected plasma proteins in young (6 months) or old (24 months) rats fed lifelong with two experimental diets enriched in either sunflower or virgin olive oil. After the depletion of the most abundant proteins, levels of less abundant proteins were studied using two-dimensional electrophoresis and mass spectrometry. Our results showed that compared with the sunflower oil diet, the virgin olive oil diet induced significant decreases of plasma levels of acute phase proteins such as inter-alpha inhibitor H4P heavy chain (at 6 months), hemopexin precursor (at 6 and 24 months), preprohaptoglobin precursor (at 6 and 24 months), and α-2-HS glycoprotein (at 6 and 24 months); antioxidant proteins such as type II peroxiredoxin (at 24 months); proteins related with coagulation such as fibrinogen γ-chain precursor (at 24 months), T-kininogen 1 precursor (at 6 and 24 months), and apolipoprotein H (at 6 and 24 months); or with lipid metabolism and transport such as apolipoprotein E (at 6 and 24 months) and apolipoprotein A-IV (at 24 months). The same diet increased the levels of apolipoprotein A-1 (at 6 and 24 months), diminishing in general the changes that occurred with age. Our unbiased analysis reinforces the beneficial role of a diet rich in virgin olive oil compared with a diet rich in sunflower oil, modulating inflammation, homeostasis, oxidative stress, and cardiovascular risk during aging.

Molecular cloning of liver Wap65 cDNA in ayu (Plecoglossus altivelis) and mRNA expression changes following Listonella anguillarum infection.

The teleost warm temperature acclimation related 65 kDa protein (Wap65) is a plasma glycoprotein with the potential roles in heat adaptation, heme recycling, immune response and copper metabolism. It is most homologous to the mammalian hemopexin, which is the plasma transporter of heme. A full-length cDNA clone of the Wap65 gene, 1,534 bp in size, was isolated from the fish ayu (Plecoglossus altivelis). Its deduced amino acid sequence of 439 residues had 60.4-65.4% and 38.3-47.3% identical to fish Wap65-2-type and Wap65-1-type sequences, respectively. In phylogenetic analysis, aWap65 grouped tightly with those fish Wap65-2-type sequences. In healthy control fish, the highest mRNA signal for aWap65 was from the liver, moderately high in brain and gill, and but weaker in spleen, kidney, muscle, heart and intestine. In Listonella anguillarum-infected fish, aWap65 transcripts were significantly increased in liver, while no obvious changes in other tissues at 12 hpi. However, aWap65 transcripts were significantly increased in various tissues at 24 hpi when hemolysis developing, suggesting that aWap65 might be involved in the immune response of ayu.

Hemin toxicity: a preventable source of brain damage following hemorrhagic stroke.

Hemorrhagic stroke is a common cause of permanent brain damage, with a significant amount of the damage occurring in the weeks following a stroke. This secondary damage is partly due to the toxic effects of hemin, a breakdown product of hemoglobin. The serum proteins hemopexin and albumin can bind hemin, but these natural defenses are insufficient to cope with the extremely high amounts of hemin (10 mM) that can potentially be liberated from hemoglobin in a hematoma. The present review discusses how hemin gets into brain cells, and examines the multiple routes through which hemin can be toxic. These include the release of redox-active iron, the depletion of cellular stores of NADPH and glutathione, the production of superoxide and hydroxyl radicals, and the peroxidation of membrane lipids. Important gaps are revealed in contemporary knowledge about the metabolism of hemin by brain cells, particularly regarding how hemin interacts with hydrogen peroxide. Strategies currently being developed for the reduction of hemin toxicity after hemorrhagic stroke include chelation therapy, antioxidant therapy and the modulation of heme oxygenase activity. Future strategies may be directed at preventing the uptake of hemin into brain cells to limit the opportunity for toxic interactions.

Dietary ß-hydroxy-ß-methylbutyrate supplementation influences performance differently after immunization in broiler chickens.

Dietary addition of the leucine metabolite ß-hydroxy-ß-methylbutyrate (HMB) promotes growth in various species. In addition, HMB is described to enhance immune responses which might be associated with metabolic costs. We elaborated further on the role of HMB in growth, metabolism and immunity of meat-type chickens using the following parameters: zootechnical performance, blood chemistry and a specific immune responses after immunization with a human serum albumin (HSA)/Freund's (in) complete adjuvant combination. The chickens received commercial feeds either unsupplemented or supplemented with 300 mg HMB/kg feed. ß-hydroxy-ß-methylbutyrate-supplemented chickens were significantly heavier at 2 weeks of age but this difference was attenuated at later ages. Compared with their unsupplemented controls, cumulative feed conversion was significantly lower in HMB-supplemented chickens. There were no differences in blood chemistry between both dietary treatments. After immunization, HMB significantly attenuated the acute phase protein response at day 1 of post-immunization compared with that of their unsupplemented counterparts. After day 7 post-immunization, body weight gain of the immuno-challenged HMB-supplemented chickens was significantly depressed, but their specific anti-HSA IgG response was significantly enhanced compared with that of their immuno-challenged unsupplemented counterparts. The underlying mechanisms and signalling pathways for these phenomena need to be elucidated. Nevertheless, we are able to conclude that HMB is beneficial for performance under normal circumstances. On the other hand, HMB stimulates the immune response after an immunological challenge, though at the cost of reduced growth.

Proteomics of S-(1, 2-dichlorovinyl)-L-cysteine-induced acute renal failure and autoprotection in mice.

Previous studies (Vaidya VS, Shankar K, Lock EA, Bucci TJ, Mehendale HM. Toxicol Sci 74: 215-227, 2003; Korrapati MC, Lock EA, Mehendale HM. Am J Physiol Renal Physiol 289: F175-F185, 2005; Korrapati MC, Chilakapati J, Lock EA, Latendresse JR, Warbritton A, Mehendale HM. Am J Physiol Renal Physiol 291: F439-F455, 2006) demonstrated that renal repair stimulated by a low dose of S-(1,2-dichlorovinyl)l-cysteine (DCVC; 15 mg/kg i.p.) 72 h before administration of a normally lethal dose (75 mg/kg i.p.) protects mice from acute renal failure (ARF) and death (autoprotection). The present study identified the proteins indicative of DCVC-induced ARF and autoprotection in male Swiss Webster mice. Renal dysfunction and injury were assessed by plasma creatinine and histopathology, respectively. Whole-kidney homogenates were run on two-dimensional gel electrophoresis gels, and the expression of 18 common proteins was maximally changed (> or =10-fold) in all the treatment groups and they were conclusively identified by liquid chromatography tandem mass spectrometry. These proteins were mildly downregulated after low dose alone and in autoprotected mice in contrast to severe downregulation with high dose alone. Glucose-regulated protein 75 and proteasome alpha-subunit type 1 were further investigated by immunohistochemistry for their localization in the kidneys of all the groups. These proteins were substantially higher in the proximal convoluted tubular epithelial cells in the low-dose and autoprotected groups compared with high-dose alone group. Proteins involved in energetics were downregulated in all the three groups of mice, leading to a compromise in cellular energy. However, energy is recovered completely in low-dose and autoprotected mice. This study provides the first report on proteomics of DCVC-induced ARF and autoprotection in mice and reflects the application of proteomics in mechanistic studies as well as biomarker development in a variety of toxicological paradigms.

Dietary L-carnitine supplementation enhances the lipopolysaccharide-induced acute phase protein response in broiler chickens.

The objective of the present study was to evaluate the potential effects of dietary L-carnitine supplementation on acute phase protein response upon a lipopolysaccharide (LPS) challenge of male broiler chickens receiving a commercial broiler diet supplemented with 15 or 100 mg L-carnitine/kg or an unsupplemented (control) diet from 14 days of age onwards. At 28 days of age, eight chickens per dietary treatment were weighed and subcutaneously injected with 300 microg LPS from E. coli (100 microg LPS/ml saline) or 3 ml saline (unsupplemented group only). During the next 10 days, blood samples were taken repeatedly and analysed for their hemopexin (HX) and alpha-1 acid glycoprotein (AGP) levels. Extra dietary L-carnitine did not affect broiler performance. At day 1 postinjection, plasma HX and AGP levels were significantly increased in all treatment groups. However, the elevations in circulating HX and AGP levels were more pronounced in the L-carnitine supplemented chickens, especially in the 100mg L-carnitine group. It is concluded that extra L-carnitine in the diet of broiler chickens enhances or advances the acute phase protein response. The exact mode of action needs to be elucidated but seems to be consistent with a glucocorticoid mimicking effect.

Identification of hemopexin as a GH-regulated gene.

A cDNA library from the liver of a growth hormone (GH)-treated hypophysectomized rat was constructed and screened for GH-inducible genes (GIGs). Three cDNAs specific for putative GIG mRNAs (GIG-3, -7 and -12) were isolated and, when sequenced, were found to be homologous to portions of rat hemopexin, a Class 2 acute-phase gene. Hemopexin is an essential heme scavenger produced primarily in the liver, which upon binding to free heme, transports it to the liver where the heme iron is re-utilized. Hemopexin has not been previously described as being GH-responsive. GIG-3 and GIG-12 encode overlapping portions of the entire coding sequence starting within a few hundred base pairs from the 5' end of the hemopexin mRNA, and GIG-7 encodes the 3'-most end of the hemopexin mRNA. Northern analysis and ribonuclease protection assays of RNA from livers of control rats using the cDNA probes demonstrated a major transcript of approximately 2.0 kb. The hemopexin mRNA was low or undetectable in livers of hypophysectomized rats. Daily treatment with bovine growth hormone (bGH) for 10 days restored hemopexin mRNA to levels comparable or greater than that of intact rats. GH-dependence in cultured rat H4IIE hepatoma cells was then examined. Using hemopexin cDNA probes (GIG-3, -7, and -12) we identified a mRNA on Northern blots, which increased in concentration following bGH, compared with untreated cells. When measured by ribonuclease protection assay, a maximal increase in hemopexin mRNA concentration was obtained following 4-6 h of bGH administration. We conclude that hemopexin is a GH-inducible gene in rat liver in vivo and in cultured rat hepatoma cells.

Abnormal iron parameters in the pregnancy syndrome preeclampsia.

OBJECTIVE: Our purpose was to investigate iron status parameters in preeclampsia with a view to exploring their possible contribution to the etiology. STUDY DESIGN: In prepared serum samples from 40 preeclamptic women and matched pregnant control subjects at the John Radcliffe Hospital, Oxford, a number of iron status parameters were measured. Statistical analysis was by the Wilcoxon signed rank test and linear regression. RESULTS: Serum iron concentration, ferritin, and percent saturation of transferrin were significantly higher in the preeclamptic patients than in control subjects, whereas unsaturated iron-binding capacity and apotransferrin levels were significantly lower. No difference was found in hemopexin concentrations in the two groups. Gestational age at the time of sampling was correlated (positively) with only two parameters, total and unsaturated iron-binding capacity, but only in the preeclampsia group. Eighteen percent of preeclamptic subjects had percent transferrin saturation levels in the region associated with iron overload. CONCLUSION: Released iron species in preeclampsia may contribute to the etiology and will exacerbate lipid peroxidation and endothelial cell injury. Given the high prevalence of heterozygosity for hemochromatosis with the associated reduced ability to exclude ingested iron, it would seem inadvisable, in the absence of evidence of iron deficiency, to give iron supplements to pregnant women at high risk for preeclampsia.

The kinetics of hemopexin and alpha1-acid glycoprotein levels induced by injection of inflammatory agents in chickens.

The acute phase response to inflammation induces changes in the secretion of hepatic proteins. To examine the time course of an acute phase protein response in broiler chickens, the plasma levels of hemopexin (HX) and alpha1-acid glycoprotein (AGP) and liver HX mRNA were measured at various time points from 3 hr to 336 hr after an intraabdominal injection of either lipopolysaccharide (LPS), complete Freund's adjuvant, incomplete Freund's adjuvant, phytohemagglutin, or mineral oil. Uninjected chicks served as controls. The accumulation of liver HX mRNA began within 3 hr of stimulation and peaked at 12 hr. Relative to control levels, plasma HX and AGP levels increased by 6-12 hr postchallenge and peaked at 24 hr. Complete Freund's adjuvant and LPS treatments induced the greatest increase in plasma HX (threefold; P < 0.05). Plasma levels of HX and AGP returned to control levels at 336 and 168 hr postinjection, respectively. A second experiment demonstrated that turpentine induced a similar AGP response as LPS and that albumin is a negative acute phase protein. The results suggest that plasma levels of HX or AGP could be used as an indicator of the systemic component of a local inflammatory response in chickens.

Dietary fish oil or lofrin, a 5-lipoxygenase inhibitor, decrease the growth-suppressing effects of coccidiosis in broiler chicks.

Broiler chicks were fed a diet containing 4% of either corn oil or fish oil from 3 to 14 d of age. From Days 15 to 23, half of the chicks in each dietary treatment were fed Lofrin (an experimental 5-lipoxygenase inhibitor) at 33 micrograms/kg feed. The remaining chicks within each dietary treatment were the untreated controls. At 24 d of age, half of the chicks within each diet-Lofrin treatment group were each infected with 4.6 x 10(4) sporulated Eimeria tenella oocysts, resulting in a 2 x 2 x 2 factorial arrangement of treatments. Body weight gain, feed consumption, and feed conversion efficiency were determined throughout the study. At 27 d of age, blood, liver, and ceca were sampled. Plasma tumor necrosis factor and hemopexin, hepatic fatty acid composition, and cecal inflammatory cell infiltration were determined. Liver fatty acid composition tended to reflect that of the diet. Chicks fed fish oil had livers that were enriched in (n-3) polyunsaturated fatty acids (PUFA) at the expense of (n-6) PUFA. Chicks fed fish oil gained body weight more rapidly than those fed corn oil. Infection of chicks with Eimeria decreased body weight gain of chicks fed corn oil, but not of chicks fed fish oil. The addition of Lofrin to the corn oil diets abrogated the growth-suppressing effects of infection, although there was no Lofrin effect among chicks fed fish oil. There was a diet by Lofrin interaction in which Lofrin treatment of birds fed corn oil decreased feed consumption and increased feed conversion efficiency, but had no effect on chicks fed diets containing fish oil. Plasma hemopexin was greater, but tumor necrosis factor was lower, in chicks fed fish oil than in chicks fed corn oil. Eimeria infection significantly increased cecal inflammatory cell infiltration across all dietary treatments. There were no clear relationships between growth rate or efficiency and the severity of the inflammatory response to Eimeria infection, as indicated by hemopexin levels and cecal inflammatory scores. These results indicate that Lofrin or fish oil, both of which modify eicosanoid metabolism, attenuate the growth-depressing effects of an Eimeria tenella infection.

Role of heme-hemopexin in human T-lymphocyte proliferation.

Heme-hemopexin supports and stimulates proliferation of human acute T-lymphoblastic (MOLT-3) cells, suggesting the participation of heme in cell growth and division. MOLT-3 cells express approximately 58,000 hemopexin receptors per cell (apparent Kd 20 nM), of which about 20% are on the cell surface. Binding is dose- and temperature-dependent, and growth in serum-free IMDM medium is stimulated by 100-1000 nM heme-hemopexin, consistent with the high affinity of the receptor for hemopexin, and maximal growth is seen in response to 500 nM complex. Growth was similar in defined minimal medium supplemented with either low concentrations of heme-hemopexin or iron-transferrin, and either of these complexes were about 80% as effective as a serum supplement. Heme-hemopexin, but not apo-hemopexin, reversed the growth inhibition caused by desferrioxamine showing that heme-iron derived from heme catabolism is used for cell growth. Cobalt-protoporphyrin (CoPP)-hemopexin, which binds to the receptor but is not transported intracellularly [Smith et al., (1993) J. Biol. Chem. 268, 7365], also stimulated cell proliferation in serum-free IMDM but did not "rescue" the cells from desferrioxamine. Furthermore, CoPP-hemopexin effectively competed for the hemopexin receptor with heme-hemopexin and diminished its growth stimulatory effects. In addition, protein kinase C (PKC) is translocated to the plasma membrane within 5 min after heme-hemopexin is added to the medium, reaches maximum activity within 5-10 min, and declines to unstimulated levels by 30 min. Heme-hemopexin and CoPP-hemopexin both augmented MOLT-3 cell growth stimulated by serum. Thus, heme-hemopexin not only functions as an iron source for T-cells but occupancy of the hemopexin receptor itself triggers signaling pathway(s) involved in the regulation of cell growth. The stimulation of growth of human T-lymphocytes by heme-hemopexin is likely to be a physiologically relevant mechanism at sites of injury, infection, and inflammation.

Photodynamic killing of retinoblastoma cells with hematoporphyrin and light.

Hematoporphyrin and white light exerted a lethal effect on two cell lines of retinoblastoma cells. The lowest values of dye concentration and light exposure capable of killing an entire cell population were, respectively, 2 X 10(-5) M and 6 min exposure to an irradiance of 6.0 microwatts/sq mm. Cells exposed to light in the presence of the dye did not require lengthy incubation periods but were rapidly killed with increasing periods of light exposure. Cells washed free of the dye, however, required a minimum sensitizing period of 3.5 hr to achieve a value close to 100% cell death. An inhibitor of this photodynamic process was demonstrated in normal serum. When the concentration of either fetal calf serum, rabbit serum, or rabbit plasma was increased to 25% from a standard 10%, there was as much as a 100-fold greater requirement of hematoporphyrin concentration to produce the same lethal response. The suggested explanation for this phenomenon is a porphyrin-binding plasma factor, hemopexin, which is a natural beta-glycoprotein believed to be responsible for the transport of circulating porphyrins to the liver for their elimination. Retinoblastoma cells grow in suspension and thereby provide an excellent tool for study of photosensitive dyes, especially in the case of the rapidly growing Y79 cell line.

[Intraoperative autotransfusion and haemolysis (author's transl)]. / Erste Erfahrungen mit der maschinellen intraoperativen Autotransfusion. Die Haemolyse.

In 29 patients (12 vascular and 17 trauma cases) receiving autotransfusion the effects of haemolysis caused by the Bentley ATS-system were examined. The following parameters were monitored: 1. Overall haemolysis rate and fractions in serum and urine, --2. Total and direct bilirubin in all patients with or without preexisting icterus, --3. Plasma-proteins: Albumen, haptoglobin, haemopexin, transferrin and C3-activator. --In both groups extremely high rates of free haemoglobin in serum were found in some cases. The peak of haemoglobinuria was observed several hours after the autotransfusion or at the end of the operation. The different plasma proteins showed increased activity to cope with haemolysis within the first 24 h. After one week they still showed overshooting levels in some cases which permitted conclusions concerning the extent of the reactions. The transformation of free haemoglobin in bilirubin has to be strongly suspected. The changes of the parameters were not in relation to the volume of autotransfusion. No irreversible complications due to haemolysis caused by the autotransfusion systems were observed.