Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies

Cryptococcal meningitis occurs in 6 to 8% of human immunodeficiency virus-infected individuals. Despite the availability of powerful antifungal agents that are active against Cryptococcus neoformans, these drugs generally fail to cure cryptococcal infections in immunocompromised hosts. Alternative approaches to prevention and therapy of cryptococcosis are urgently needed. Complement promotes phagocytosis of C. neoformans, but human antibodies to cryptococcal capsular polysaccharide have not been shown to function as complement-independent opsonins. The goal of our studies was to characterize the in vitro biological function of human antibodies to glucuronoxylomannan (GXM) from individuals immunized with a GXM-tetanus toxoid (GXM-TT) vaccine. We studied sera from nine vaccinees that manifested good serologic responses to GXM-TT. The results indicate that GXM-TT-elicited antibodies promote phagocytosis of C. neoformans by both murine J774 cells and human peripheral blood mononuclear cells (PBMCs). The two sera with the highest titers of anti-GXM immunoglobulin G2 antibodies were the most opsonic. When PBMC Fc gamma RIIa receptors were blocked, a 75% decrease in phagocytosis occurred following incubation of the PBMCs with C. neoformans opsonized with these sera. Our data indicate that, in the absence of complement, human anti-GXM-TT antibodies are opsonic and that antibodies of the immunoglobulin G2 isotype are effective opsonins.     

Opsonin requirements for phagocytosis of Enterococcus spp. by human polymorphonuclear leukocytes

BACKGROUND: Interaction of Enterococcus spp. and host defense mechanisms is not well known. Opsonic requirements of Enterococcus faecalis and Enterococcus faecium to be phagocyted by human polymorphonuclear leukocytes (PMN) were evaluated. METHODS: Twenty strains (10 E. faecalis and 10 E. faecium) were studied. Phagocytosis was determined by a radiometric assay. Bacterial cells were labelled with 3H-adenine and opsonized with: a) 10% of human pool sera (HPS); b) 10% of decomplemented HPS, and c) albumin and fibronectin. RESULTS: Phagocytosis of Enterococcus spp. by PMN in the presence of HPS was significantly higher than that in the absence of opsonins. The phagocytosis of E. faecium was higher than that of E. faecalis. A strain-dependent effect of complement in the phagocytosis of Enterococcus spp. was observed. Neither albumin nor fibronectin showed an opsonic activity on Enterococcus spp. CONCLUSIONS: A great heterogeneity in the opsonic requirements of Enterococcus spp. was observed. Serum opsonins show a critical role in the phagocytosis of E. faecalis and E. faecium by PMN, this effect being more relevant with E. faecium. A strain-dependent opsonic activity of complement was observed.     

Locus coeruleus lesions decrease norepinephrine input into the medial preoptic area and medial basal hypothalamus and block the LH, FSH and prolactin preovulatory surge

Phagocytosis is a fundamental process in innate resistance to infection. We have used the pathogenic yeast Cryptococcus neoformans to study the interaction of this encapsulated organism with murine macrophages in vitro. In the absence of exogenous opsonins the encapsulated yeast is almost totally resistant to ingestion by murine macrophages. Owing to its ability to activate the alternative complement pathway, the anti-phagocytic properties of the polysaccharide capsule can be partially overcome following opsonization in vitro with non-immune mouse serum and subsequent phagocytosis via complement receptors. Here, we demonstrate the importance of the complement receptor type 3 (CR3) in in vitro phagocytosis of the yeast and in in vivo resistance to infection. In vitro, 70% of a population of resident murine macrophages are able to ingest C. neoformans and then only inefficiently (1-2 organisms per cell). Previously we have shown that tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) efficiently enhance ingestion of serum-opsonized encapsulated C. neoformans, and we now show that the cytokines convert a population of resident macrophages to a state where all the cells are competent for ingestion of large numbers of yeasts (6-8 per cell). We also show that these cytokines have a direct effect on CR3, as enhanced levels of complement-opsonized sheep red blood cells (EIgMC) bind to macrophages activated in this way. However, cytokines that have previously been shown to enhance phagocytosis of EIgMC have no effect on ingestion of encapsulated C. neoformans. These results demonstrate that the cytokines regulating CR3-dependent ingestion of C. neoformans are different to those regulating ingestion of EIgMC and reinforce the importance of studying pathogens rather than inert ligands in understanding the regulation of phagocytosis.     

Role of CR4 in Mycobacterium tuberculosis-human macrophages binding and signal transduction in the absence of serum

The beta2 integrin CR4 is involved in Mycobacterium tuberculosis phagocytosis by human mononuclear phagocytes through the opsonin C3bi. In this study, we demonstrate that M. tuberculosis can bind directly to monocyte-derived macrophages via CR4 in the absence of any opsonins. CR4-transfected CHO cells gave similar results, suggesting recognition by CR4 of bacterial structure. Furthermore, binding of M. tuberculosis transduced a potent signal, resulting in tyrosine phosphorylation of macrophage proteins, which was in part mediated by CR4.     

A role for scavenger receptors in phagocytosis of protein-coated particles in rainbow trout head kidney macrophages

In macrophages of higher vertebrates, Fc receptors and receptors for complement and other serum factors, are generally known to enhance the phagocytic process. In lower vertebrates like salmonid fishes, none of these or other phagocytic receptors have been thoroughly characterized. The purpose of this study was to elucidate to what extent these and other receptors are involved in the process of phagocytosis in rainbow trout (Oncorhynchus mykiss) head kidney macrophages. We used tosyl activated, paramagnetic dynabeads (2.8 microm in diameter), specifically coated with 125I labeled Atlantic salmon (Salmo salar) IgM or bovine serum albumin (BSA) as phagocytic probes. The effect of complement opsonization was also investigated by incubating the beads in serum. Our results indicate that neither the Fc- nor the complement-receptor(s) were important for phagocytosis of these beads. Our data support the idea that scavenger receptors are involved in phagocytosis in rainbow trout head kidney macrophages, as the use of a competitive scavenger receptor ligand extensively decreased degradation of the labeled protein coat on the beads.     

Response of odontoblast-like cells to hydroxyapatite ceramic granules

Previous studies have suggested that, in vivo, activated T lymphocytes and neutrophils are important in immunity to nontypable Haemophilus influenzae. We now extend this work by showing that neutrophils pretreated with products of activated T lymphocytes or activated macrophages show significantly enhanced killing of nontypable H. influenzae. Lymphotoxin, a product of activated T lymphocytes, significantly enhanced the neutrophil-mediated killing of nontypable H. influenzae, and tumor necrosis factor, produced by activated T lymphocytes as well as macrophages stimulated by activated T lymphocytes, also significantly increased the bactericidal activity of neutrophils. These cytokine-induced effects were seen with short pretreatment times of neutrophils and were maximal by 30 min. The killing of H. influenzae by neutrophils required the presence of heat-labile opsonins. In the absence of these opsonins, both tumor necrosis factor and lymphotoxin were unable to promote the killing of the bacteria by neutrophils. Furthermore, the results showed that tumor necrosis factor-primed neutrophils displayed significantly increased expression of CR3 and CR4 that was associated with increased phagocytosis of complement-opsonized nontypable H. influenzae. These cytokines may play an important role in immunity toward nontypable H. influenzae by stimulating neutrophil bactericidal activity.     

Enhanced hepatic uptake of liposomes through complement activation depending on the size of liposomes

The objective of this study was to differentiate the roles of opsonins and phagocytic cells in the size-dependent hepatic uptake of liposomes in the submicron region. The extent of opsonization decreased with the decrease in size of liposomes (from 800 to 200 nm in diameter) and no enhancement of uptake was observed at 200 nm. There was no effect of liposome size on the uptake of unopsonized liposomes. Serum was pretreated with empty liposomes of each size and its opsonic activity was measured in the perfused liver. The small liposomes could not consume the opsonic activity, while the larger ones did so substantially. These results suggest that opsonins bind to liposomes depending on the size of liposomes and phagocytic cells take up liposomes in proportion to the extent of opsonization. Size-dependent liposome degradation in serum was also found, which was consistent with the size-dependent complement activation, because liposomes with this composition have been shown to be degraded by complement. The mechanism of opsonization was examined by treating serum at 56 degrees C for 30 min or with anti-C3 antiserum. Since both treatments inhibited the opsonic activity, the hepatic uptake of liposomes is considered to occur via complement receptor. In conclusion, the size of liposomes affected complement recognition, and the liposomes were taken up by the liver depending on the extent of opsonization.     

IgE levels in cord blood in an area of the Veneto region

BACKGROUND: Cord blood IgE levels have been studied as a possible marker of allergy in infants but few studies are available in our Region. The aim of this paper was to test IgE levels in cord blood of 60 consecutive newborns in a restricted area of Veneto, to correlate cord blood IgE levels with family history of allergy and to verify the risk of contamination from mother's blood. METHODS: Cord blood was obtained from 60 consecutive newborns. Immunoglobulin levels (IgG, IgA, IgM, and IgE) were measured in cord blood of newborns and in serum of all mothers. Family history for allergy was previously investigated from the mothers. RESULTS: IgE were detectable in cord blood of 5 newborns but only 2 of them had positive family history for allergy which was pointed out in 11/60. In one of these cases the contamination of sample from mother's blood was postulated. IgG levels in newborn cord blood were higher than in mothers' blood and it was not related with IgE levels or other investigated factors. CONCLUSIONS: Only 6.6% of newborns in a restricted area of Veneto region have detectable IgE in cord blood whereas 18.3% of them have positive family history for allergy. Measurable levels of IgE in cord blood are not related with positive family history of allergy and are rarely influenced by mothers' blood contamination.     

Complement, opsonins, and the immune response to bacterial infection in burned patients

Studies were performed to evaluate complement, opsonins, and the immune response to bacterial infection in burned patients. Concentrations and functional acitivities of components of the classical and alternative complement pathways were measured in the sera of four septic, two bacteremic, and four nonseptic burned patients. In addition, heat-labile and heat-stable opsonic activity and agglutinin titers directed against the infecting bacterial strains were measured in the sera of the four septic patients and in an additional group of 11 septic burned patients with abnormal complement profiles. Functional activity of the alternative complement pathway and the concentration of properdin were shown to be persistently decreased during eight weeks postburn in the septic, bacteremic, and nonseptic burned patients; reduced classical pathway activity was demonstrated during the initial postburn period only in the septic patients. Two of the 15 septic patients had decreased heat-labile serum opsonic activity for their infecting bacterial strains, which occurred only during the initial postburn period. Heat-stable opsonins and agglutinin titers in the patients' sera directed against the infecting bacterial strains were equivalent to those in normal human sera, except for the agglutinin titers to Streptococcus faecalis which were increased in the patients' sera in comparison to the normal sera. These results indicate that the multiple complement abnormalities which occur in septic burned patients do not predispose these patients to bacterial infection by decreasing serum opsonic activity. Moreover, heat-stable immune IgG antibodies are not produced during septicemia which facilitate opsonization of the infecting bacterial strains in the absence of an intact complement system.     

Influence of neonatal idiopathic respiratory distress syndrome on serum enzyme activities in premature healthy and asphyxiated newborns

Elevated serum aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), and hydroxybutyrate dehydrogenase (HBDH) activities are highly predictive for the development of hypoxia-related neurologic disorders in asphyxiated newborns. Little is known about the influence of the idiopathic respiratory distress syndrome (IRDS) on the serum profile of these enzymes. In a prospective study we measured ASAT, LDH, and HBDH activities in asphyxiated newborns with (n = 12) and without (n = 12) IRDS as well as non-asphyxiated newborns with IRDS (n = 16). Blood samples were taken serially at five fixed times: 0 (cord), 12, 24, 72, and 144 hours postpartum. Whereas both study groups of asphyxiated newborns showed significantly elevated enzyme activities as compared with the non-asphyxiated controls, the fundamental influence of perinatal hypoxic-ischemic events on neonatal serum enzyme profiles and activities of all three enzymes was not significantly altered by the development of IRDS. Therefore, the predictive value of these enzymes for the development of neurologic disorders in asphyxiated newborns is not adversely affected by the development of an immediately and effectively treated IRDS. It is concluded that elevated ASAT, LDH, and HBDH activities can be used as predictors for neurologic disorders in asphyxiated newborns even in the presence of IRDS.     

Extracellular and bacterial factors influencing staphylococcal phagocytosis and killing by human polymorphonuclear leukocytes

Extracellular and bacterial factors that influence the phagocytosis and killing of staphylococci by human polymorphonuclear leukocytes have been studied. Staphylococcus epidermidis strains were, in general, more rapidly phagocytized than were S. aureus strains. However, two strains of S. epidermidis had a very slow rate of ingestion. Although the rate of phagocytosis of S. aureus Wood 46 was greater than that of S. aureus 502A, the Wood 46 strain was more difficult to kill. Serum was essential for phagocytosis of both S. aureus and S. epidermidis. The opsonic titer of pooled serum was similar for S. aureus and S. epidermidis. In normal pooled serum, heat-labile factors were more important for effective phagocytosis than they were in immune serum. Although a saturation point for ingestion was reached, the percentage of ingested bacteria that remained alive within the leukocyte remained relatively fixed. Heat-killed and live staphylococci were igested in a similar fashion. The rate of phagocytosis was greatly reduced at 41 degrees C.     

Congenital stricture of male urethra

It was intendet to characterize the isoenzymes of serum alkaline phosphatase in healthy infants. Quantitative estimation of the activity of serum alkaline phosphatase resulted in relatively low values in the newborns and a distinct increase during the first three months of live. As a rule most of the newborns appeared to present one uniform isoenzyme fraction, which was comparable to the isoenzyme of bone origin of the adult. Some of the newborns already exhibited another less marked isoenzyme, the isoenzyme of liver origin. During babyhood this isoenzyme becomes more and more distinct. In most cases this development had become perfect at the end of the first year. Liver tissue of newborns apparently does not synthesize the same liver isoenzyme as liver tissue of adults.     

Vitronectin is responsible for serum-stimulated uptake of rod outer segments by cultured retinal pigment epithelial cells

PURPOSE: To examine whether the vitronectin (VN) in serum is responsible for the serum stimulation of phagocytosis in the rod outer segment (ROS) by cultured retinal pigment epithelial (RPE) cells. METHODS: Vitronectin was removed from fetal bovine serum by heparin-agarose affinity chromatography. Concentrations in normal and depleted serum were determined by enzyme-linked immunosorbent assay, using a polyclonal antibody against bovine VN and commercially prepared human VN as a standard. A monoclonal antibody against human alpha v beta 5 was used in localization and in blocking experiments. Rod outer segment phagocytosis was measured using a flow cytometric assay. RESULTS: Affinity chromatography removed 95% of the VN from serum as determined by enzyme-linked immunosorbent assay. Vitronectin-depleted serum did not stimulate ROS phagocytosis by RPE cells. Commercially prepared VN added to serum-free medium stimulated ROS phagocytosis in a dose-dependent manner. Pretreatment of RPE cells with an antibody against alpha v beta 5, an integrin receptor for VN, had no effect on phagocytosis in the absence of serum but completely blocked the serum stimulation of ROS phagocytosis. Antibody against alpha v beta 5 demonstrated a variable labeling pattern on the cultured RPE cell surface with morphologically distinct cell clusters exhibiting less labeling. Those cell clusters exhibiting less receptor labeling also showed less uptake of fluorescent-labeled ROS. CONCLUSIONS: Vitronectin is the component responsible for serum stimulation of ROS uptake, and this uptake appears to be mediated by an alpha v beta 5 integrin. Although clearly important in vitro, a role for VN in ROS uptake by RPE cells in situ remains to be determined.     

Bispecific-armed, interferon gamma-primed macrophage-mediated phagocytosis of malignant non-Hodgkin's lymphoma

To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony-stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.     

Onset of cell proliferation in the shortened gut: growth after subtotal colectomy

Serum IgE levels were studied in 2 groups of children with a family history of atopic disease, 30 in whom the mother only and 38 in whom both parents had atopic disease. IgE antibodies were determined with Phadebas RAST Test and serum IgE with Phadebas IgE Test and Phadebas PRIST at 0, 3, 9, 12 and 18 months of age. There was no correlation between the serum IgE levels in mothers and their newborns. RAST tests were frequently positive in maternal sera but no positive RAST test was found in the newborns. Obvious and probable atopic disease developed during the observation period in 42.1% of the children with a double family history of atopic disease. In 75% of these the serum IgE level was above the upper limit of normal on an average 6 months before the onset of atopic symptoms. An elevated IgE level without atopic symptoms during the observation period occurred in only one child. It is concluded that the serum IgE in newborns seems to be of foetal origin and that the determination of serum IgE in infants is of value in predicting atopic allergy.     

Recognition and clearance of liposomes containing phosphatidylserine are mediated by serum opsonin

Liver uptake of liposomes containing phosphatidylserine was studied in a single pass liver perfusion system and found to be serum dependent. The effectiveness of serum in mediating liposome uptake by the liver depends on liposomes size. Large liposomes appeared to be opsonized more efficiently and, therefore, taken up more by the liver than the smaller ones. The effects of liposomes size on liver uptake did not occur in the absence of serum. Treatment of serum at 56 degrees C for 30 min abolished the serum activity, suggesting the involvement of complement components. Inhibition of the hemolytic activity of complement through the alternative pathway by PS-containing liposomes suggests that components in this pathway are responsible for liposome opsonization. Liposomes containing phosphatidic acid, phosphatidylglycerol, and dicetyl phosphate compete in different degrees for serum components which mediate the liver uptake of PS-containing liposomes. These results suggest that the opsonization of liposomes by serum opsonins are the determining factors for the recognition and clearance of liposomes by the RES. Complement components are most likely involved in this process. The results presented here are relevant to the use of liposomes as drug delivery vehicle in vivo and to the PS-mediated clearance of red blood cells from the blood circulation.     

Controlled gentamycin treatment in prematures and newborns (author's transl)

On the basis of our experience with 50 prematures and newborns and 200 serum level analyses, Gentamycin doses of larger than or equal to 5 mg/kg body weight/day (depending on the pathogenic sensitivity) is necessary in life-threatening infections to obtain a serum level of 4-5 mug/ml. Our findings in this regard correspond with the results of other authors with comparable test systems. However, we found that in ill prematures and newborns, an accumulation inside of 7-10 days is the rule rather than the exception. With increasing length of therapy, Gentamycin treatment should be discontinued if the general condition and/or kidney function worsens. Treatment should be discontinued until the serum concentration has been established or a drop in serum creatine occurs. If there is no possibility of conducting control checks, Gentamycin therapy should not extend more than one week and should not exceed 2 mg/kg body weight/day for prematures and newborns. The available tests for determinin aminoglycosides in the serum must be simplified and standardized so that they can be more readily used. Such a simplification and standardization is possible as we have shown. In this way a basis is established for future follow-up checks and the risk of side effects in the inner ear and kidneys is reduced. The same is true for the new aminoglycosides Tobramycin, Amicacin, Sisomycin.     

Further evidence for the presence of cannabinoid CB1 receptors in mouse vas deferens

Basophil granulocytes and their mediators are involved in the pathogenesis of allergic inflammation. We evaluated basophil count, blood histamine content, eosinophil count and serum total IgE levels in one hundred-thirteen healthy newborns at birth. 108 children were prospectively studied with a follow-up to 18 months of age for development of topic disorders. No difference was found in newborns with biparental family history of atopy (FHA) in comparison with newborns with monoparental FHA and with newborns without FHA. Children who developed atopic disorders had neonatal basophil count higher than those who did not develop atopic symptoms (p = 0.03). No significant correlation was found between basophil and eosinophil counts (r = 0.013), between basophil count and serum total IgE levels (r = 0.012) and between basophil count and blood histamine content. Positive predictive value and sensitivity of basophil count for allergy up to 18 months of age was respectively only 33% and 27%. Our data indicate that an increased basophil count at birth is not associated with FHA and is not a good predictive marker of atopy.     

Vitamin E and C levels in infants during the first year of life

The considerably lower vitamin E level found in cord blood and in newborns at birth than those found in the venous blood of mothers at delivery are not yet fully explained. In a group of 217 not selected newborns, we attempted to establish the relation between vitamin E and C levels at delivery and the changes during the first year of life. The mean serum vitamin E level rose from 0.37 mg/ml at 3 days to 0.80 mg/100 ml at 6 months and to 0.72 mg/100 ml at 12 months. On the other hand vitamin C mean levels lowered from 0.93 mg/100 ml in cord blood to 0.77 mg/100 ml at 6 months and to 0.73 mg/100 ml at 12 months. The rise of vitamin E values could be explained by the early use of infant solid foods with high vitamin and mineral content and by the increase of serum lipoproteins. Except at 3 days after delivery there were no individual values of serum vitamin E below the acceptable 0.35 mg/100 ml limit. However, serum vitamin C levels compatible with a moderate risk were very often observed, i.e., in 27.1% of infants at 6 months and in 30.5% at 1 year. Thus, vitamin E intake in infants was satisfactory with the usual diet but not vitamin C for which blood levels were not adequate. In view of these findings it appears necessary to evaluate periodically the vitamin E as well as vitamin C status in the infant population.     

Phagocytosis, killing, and oxidant production by bovine monocyte-derived macrophages upon exposure to Brucella abortus strain 2308

Phagocytosis and intracellular survival of Brucella abortus, and oxidant production by monocyte-derived macrophages from ten B. abortus-naive cows were studied. Phagocytosis of bacteria opsonized with naive-autologous sera or reactor serum was significantly less than phagocytosis of bacteria opsonized with fetal bovine serum. After phagocytosis, intracellular survival of bacteria opsonized with naive-autologous or reactor sera was significantly less than survival of bacteria opsonized with fetal bovine serum. Production of oxidant by macrophages stimulated with B. abortus opsonized with naive-autologous, reactor, or fetal bovine sera was not significantly different. Although macrophages from one animal showed significantly less phagocytic activity, intracellular killing and oxidant production by macrophages from the ten individual cows toward B. abortus opsonized with naive-autologous, reactor, and fetal calf sera were homogeneous. The abilities of the macrophages to phagocytize and to kill B. abortus were not associated with each other or with oxidant production. Innate resistance or sensitivity to B. abortus was not identified in the cows based on macrophage function.