Background: Various cytokines are reportedly associated with many neonatal diseases. Asphyxia is considered to result in ischemia-reperfusion injuries and induces abnormal inflammatory responses involving excessive cytokine production. Objectives: To evaluate alteration in sera levels of various cytokines/chemokines in case of perinatal asphyxia at birth. Methods: In orderto determine the concentrations of various cytokines/chemokines in sera, we used a highly sensitive fluorescence microsphere method. We measured the concentration of 8 types of cytokines/chemokines in sera obtained from 17 cases of asphyxia, 10 normal neonates, and 6 healthy adults. Results: The concentrations of IL-6, IL-8, and IL-10 in the sera of asphyxiated neonates were higher than those in the normal neonates. Irrespective of the presence or absence of asphyxia, sera concentrations of IL-2, IL-4, IFN-γ, and TNF-α were higher in the neonates than those in the adults. The concentration of IFN-γ in the asphyxiated neonates was lower than that in the normal neonates. Sera levels of IL-10 were higher in the asphyxiated cases than those in the normal neonates. The sera levels of IL-6, IL-8, and IL-10 in asphyxiated neonates with either a poor outcome or death were higher than those without poor outcomes. Conclusions: The concentrations of various types of cytokines/chemokines were different in neonatal sera and some of them increased drastically during asphyxia. The concentration of an anti-inflammatory cytokine IL-10 was elevated in asphyxiated neonates immediately after birth, thereby suggesting that IL-10 might be associated with neuroprotective functions.

A transient myeloproliferative disorder (TMD) occurs in 10% of the infants with Down syndrome. While most cases resolve within a few months, in 20% of them TMDs are life-threatening or fatal. We encountered 4 patients with TMD, including 1 patient who died of liver failure and disseminated intravascular coagulation. Suspecting involvement of proinflammatory cytokines, we serially assayed them in patients' sera. Cytokines were significantly more abundant in patients than in controls. Interleukins 1 and 2, tumor necrosis factor alpha, interferon gamma, and granulocyte-macrophage colony-stimulating factor were greatly increased, especially in the infant who died. Sustained cytokinemia is likely to participate in TMD pathophysiology, and very high serum concentrations might predict a poor outcome.

The expression of adherent leukocyte surface molecules, L-selectin (CD62L), and Mac-1 (CD11b/CD18) in peripheral blood polymorphonuclear leukocytes (PMNs) was studied in 48 neonates and 24 healthy adults using fluorescence flow cytometry. L-selectin expression was decreased significantly in neonates, especially those with severe asphyxia (n = 10) compared with adults. Mac-1 expression was significantly increased in neonates compared with adults, but did not differ between 24 normal neonates, 14 neonates with mild asphyxia, and 10 neonates with severe asphyxia. These results suggest that the magnitude of the decrease in L-selectin expression reflects the severity of asphyxia in neonates.

To prove the mechanism of photon emission during activation of leukocytes, a model system of human polymorphonuclear leukocytes (PMNs)-opsonized zymosan (OZ)-tyrosine (or none) or myeloperoxidase (MPO)-H2O2-tyrosine was employed, and three parameters-chemiluminescence yield and intensity, a metabolite such as bityrosine (BT), and chemiluminescence spectra-were studied. With the PMN system, the luminescence was enhanced by addition of tyrosine, its analogues, or albumin, but was inhibited by hydroxyurea, superoxide dismutase (SOD), or NaN3 (an inhibitor of MPO), indicating participation of tyrosine phenoxyl radicals, O2.- and MPO in the luminescence. With the PMN-OZ-tyrosine system, chemiluminescence yield was parallel to the BT formation. These results were essentially the same as those obtained with the MPO-H2O2-tyrosine system, except that luminescence from the latter system was not inhibited by SOD. When human albumin was exposed to the MPO-H2O2 system, BT was detected after hydrolysis of the protein in the mixture. Judging from the chemiluminescence spectra of activated PMNs and the MPO-catalyzed tyrosine oxidation, at least two excited species in triplet states-one for tyrosine and another for BT-would be generated in these systems. The luminescence may originate from the reaction of tyrosine phenoxyl radicals (cation radicals) with O2.- and/or peroxidase compound III (Fe...IIIO2.-).

In order to clarify the relation between opsonic activity (OA) in newborn infants and their gestational age, we determined OA by Cypiridina luciferin analog-dependent chemiluminescence (MCLA-CL). In addition, complement C3 which plays the major role in OA, was determined by the two-dimensional immunodiffusion method. Although OA and C3 in newborn infants increased in proportion to their gestational age, the levels were significantly lower in full-term infants than in adults. In neonates with IgM levels of more than 30 mg/dL at birth, both OA and C3 levels were significantly higher than in normal neonates, yet these levels were significantly lower than in adults. These findings suggest that low opsonin activity is an important factor in neonatal susceptibility to infection. When OA is measured by chemiluminescence, MCLA-CL is the preferred technique to luminol-dependent chemiluminescence.

Copper, zinc-superoxide dismutase (Cu,Zn-SOD) activities were determined by the chemiluminescence method using the cypridina luciferin analog (MCLA) in 22 human brains from fetuses to adults. Cu,Zn-SOD activity of the cerebral cortex and white matter increased from 15% in fetuses to 50% of adult levels in neonates. The activity of the white matter was higher than that in the cortex in the fetal period, but was essentially the same as those of the cortex in the postnatal period. Cu,Zn-SOD activity in the central nervous system was highest in the spinal cord and higher in the order pons, medulla oblongata > cerebellum, midbrain, thalamus > putamen, pallidum and cerebrum. These low activities may be related to the vulnerability of cerebral cortex and white matter in premature infants.

Opsonin activity (OA) was investigated in serum samples from cord blood of 11 full-term newborn infants and 12 healthy adult volunteers by the method of luminol-dependent chemiluminescence (L-CL) and Cypiridina luciferin analog-dependent chemiluminescence (CLA-CL) together with the measurement of complement components (C3, C4, C3A and properdin). CL and complement components of cord samples were significantly lower than that of healthy adult blood. CLA-CL showed a significant correlationship to C3 and C3A. L-CL also displayed a significant correlationship to C3A but the relationship to C3 was not significant. From these results, when OA is measured by CL, CLA-CL is preferred to L-CL as a technique.

The ability of granulocytes to generate superoxide anions (O2-) and hypochlorite (OCl-) during phagocytosis was investigated using peripheral blood samples from adults and cord blood samples from neonates, using the chemiluminescence probe cypiridina luciferin analog (CLA) for O2- generation and luminol (L) for OCl- generation. OCl- generation by granulocytes was also monitored by taurine chloramine formation. The chemiluminescence probe based upon CLA was highly specific for and sensitive to O2- and could be adopted to determine O2- generation in terms of xanthine oxidase units. The CLA-dependent chemiluminescence by cord blood granulocytes was significantly higher than that by normal adult granulocytes. Taurine chloramine formation was significantly correlated with the L-dependent chemiluminescence (L-CL). Thus, the L-CL is considered to be mainly involved in OCl- generated by phagocytizing granulocytes. L-CLs by cord blood granulocytes and normal adult granulocytes were essentially the same during phagocytosis.

Iron-catalyzed oxidative stress generates reactive oxygen species in the kidney and induces oxidative damage including lipid, protein, and DNA modifications which induces renal injury and may lead to cancer. An analysis of oxidative stress dynamics by reactive oxygen species has not been performed non-invasively in real time in intact kidneys and is a significant challenge in biology and medicine. Here, I report that MCLA-800 is a near-infrared chemiluminescent probe that visualizes the dynamics of superoxide anion (O2•–) production and the upstream generation of reactive oxygen species in living rat kidneys suffering acute renal oxidative stress induced by intraperitoneal administration of iron3+-nitrilotriacetate (Fe3+-NTA) as a representative Fe3+ chelate. MCLA-800 was intravenously injected at 250 nmol/kg body weight and immediately transported to the kidneys with the emitting light dependent on O2•– production. The magnitude of O2•– production correlated with the Fe3+-NTA dose. O2•– was continuously produced in the blood stream following Fe3+-NTA injection at 0.15 mmol/kg body weight, while peak production in the renal cortex occurred at 24 h, then decreased to the background level at 72 h. This study clearly revealed the dynamics of Fe3+-NTA-mediated O2•– production in the living kidney by chemiluminescent imaging of O2•– production using MCLA-800.

Using four different digoxin kits, it was disclosed that the majority of various samples including amniotic fluid, cord blood, and serum from neonates contained substantial levels of digoxin-like immunoreactive substance. The differences in data seemed to be due to the range of epitopes which are recognized by antidigoxin antiserum. The day-to-day studies on sera serially obtained from infants at birth to 48 days old revealed that the level of the substance (0.31 +/- 0.12 ng/ml) in sera of the 1-day-old neonates rapidly declined to the level of 0.1 ng/ml by the 2nd postnatal wk and thereafter gradually declined. The immunological specificity and accuracy of the detection of digoxin-like immunoreactive substance was confirmed by a sample dilution test, a recovery test for standard digoxin, and an absorption test with antidigoxin antiserum. The amniotic fluid and cord blood also contained four to eight times more of a digitoxin-like immunoreactive substance than they did digoxin-like immunoreactive substance. A significant correlation was observed between the levels of digoxin-like immunoreactive substance and of digitoxin-like immunoreactive substance (p less than 0.01).