Treatment with rhG-CSF for osteomyelitis in a patient with p47-phox-deficient chronic granulomatous disease

A 24-year-old woman with osteomyelitis was diagnosed as having p47-phox-deficient chronic granulomatous disease (CGD). The patient showed a marked deficiency of p47-phox, which is very rare in Japan. As the clinical response to various antibiotics including sulfamethoxazole-trimethoprim was not satisfactory, we added recombinant human granulocyte colony-stimulating factor (rhG-CSF) to the treatment protocol. We report the beneficial clinical course of the patient, together with the effect of rhG-CSF on the granulocyte function, and the present report indicates that rhG-CSF is useful for the treatment of antibiotic-resistant infection in the variant type of p47-phox-defective CGD.     

Identification of a double mutation (D160V-K161E) in the p67phox gene of a chronic granulomatous disease patient

In neutrophils of a chronic granulomatous disease (CGD) patient with a lack of p67phox the mRNA for p67phox was present in normal amount and size. This mRNA was reverse transcribed, and the coding region was analyzed by single-strand conformation polymorphism analysis. Direct DNA sequencing allowed the identification of a A479-to-T and A481-to-G substitution in exon 5 of the p67phox gene resulting in a double nonconservative amino acid change 160Lys-to-Glu and 161Asp-to-Val (D160V-K161E). This defect was found in the genomic DNA of this patient in heterozygous state and does not correspond to those previously found in other cases of CGD lacking the p67phox.     

alpha-tocopherol inhibits the respiratory burst in human monocytes. Attenuation of p47(phox) membrane translocation and phosphorylation

Vitamin E (alpha-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. alpha-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O-2) production by leukocytes. The aim of this study was to examine the mechanism underlying the inhibitory effect of alpha-tocopherol on O-2 production by human monocytes. In activated monocytes O-2 is produced by the NADPH-oxidase enzyme complex. The oxidase activation elicited by phorbol myristate acetate (PMA) requires membrane translocation of several cytosolic factors. We found that in human PMA-stimulated adherent monocytes, alpha-tocopherol (but not beta-tocopherol) inhibited O-2 production in intact cells but had no effect on a membrane preparation containing activated NADPH-oxidase, suggesting that alpha-tocopherol impairs the assembly process of the enzyme complex. We showed that translocation and phosphorylation of the cytosolic factor p47(phox) were reduced in monocytes preincubated with alpha-tocopherol. We verified that the tryptic phosphopeptide map of monocyte p47(phox) was similar to that of neutrophil p47(phox), indicating that several serine residues were phosphorylated. Peptides whose phosphorylation is dependent on protein kinase C (PKC) were phosphorylated to a lesser degree when p47(phox) was immunoprecipitated from alpha-tocopherol-treated monocytes. In vitro, the activity of PKC from monocytes was inhibited by alpha-tocopherol in a specific manner compared with that of beta-tocopherol or Trolox(R). Membrane translocation of PKC was not affected. These results show that alpha-tocopherol inhibits O-2 production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47(phox) results from a decrease in PKC activity.     

Protein kinases potentially capable of catalyzing the phosphorylation of p47-phox in normal neutrophils and neutrophils of patients with chronic granulomatous disease

A procedure for uncovering novel protein kinases was used to search for enzymes in neutrophils that may catalyze the phosphorylation of the 47-Kd subunit of the NADPH oxidase system (p47-phox). This component of the oxidase can undergo phosphorylation on multiple sites. The method is based on the ability of renatured kinases to recognize exogenous substrates fixed in gels. We report that neutrophils contain several uncharacterized protein kinases that catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 through 331 of p47-phox. Some of these enzymes are strongly activated on stimulation of the cells with phorbol 12-myristate 13-acetate (PMA). The results indicate that the phosphorylation of p47-phox in neutrophils may be more complicated than previously appreciated and may involve multiple protein kinases. In addition, we have examined both the renaturable protein kinases and the properties of protein kinase C (PKC) in neutrophils from patients with chronic granulomatous disease (CGD) who are deficient in cytochrome b558. Previous studies have shown that these cells exhibit incomplete phosphorylation of p47-phox on stimulation. In this study, we were unable to detect any alterations in the renaturable protein kinases or PKC in CGD neutrophils that could explain these defects in the phosphorylation of p47-phox.     

Assembly and activation of the phagocyte NADPH oxidase. Specific interaction of the N-terminal Src homology 3 domain of p47phox with p22phox is required for activation of the NADPH oxidase

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (KD = 0.34 microM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.     

Disseminated infection due to Chrysosporium zonatum in a patient with chronic granulomatous disease and review of non-Aspergillus fungal infections in patients with this disease

BACKGROUND: Insulin-like growth factor-2 (IGF-2) is considered one of the autocrine growth factors in colorectal carcinoma. In addition, it is well known that IGF-2 is produced in the liver. However, the role of IGF-2 in liver metastasis is not yet understood clearly. METHODS: Immunohistochemical staining of IGF-2 and IGF-1 receptor (IGF-1R) was performed on tissue samples of liver metastases from 30 colorectal carcinoma patients. In situ hybridization of IGF-2 also was conducted on the same tissue samples. Furthermore, proliferating cell nuclear antigen (PCNA) was immunohistochemically stained for use as an indicator of the proliferative activity of cancer cells. RESULTS: Invasive margins of liver metastases were stained highly by both IGF-2 (70%) and IGF-1R (83%). Overexpression of IGF-2 protein and mRNA was observed in the normal liver adjacent to the tumor. The PCNA labeling indices (LIs) of the IGF-2 positive groups were significantly higher than those of the IGF-2 negative group (P < 0.0001). In addition, the PCNA LIs for the IGF-1R positive groups also were significantly higher than those for the IGF-1R negative group (P=0.0002). CONCLUSIONS: These findings suggest that hepatocyte-derived IGF-2 stimulates tumor cell proliferation by a paracrine mechanism and plays an important role in tumor progression in colorectal carcinoma patients with liver metastases.     

The role of host (endogenous) T cells in chronic graft-versus-host autoimmune disease

Chronic graft-vs-host (cGVH) disease induced by the transfer of Ia-incompatible spleen cells from one normal mouse strain (such as B6.C-H2(bm12)/KhEg (bm12)) to another (such as C57BL/6) causes an autoimmune syndrome resembling systemic lupus erythematosus (SLE). The role of host-derived T cells in this response is not obvious. Previous reports suggested that host T cells might serve to down-regulate the autoimmune syndrome. To address this issue more definitively, we used CD4 knockout (KO) or CD8KO C57BL/6 (B6) mice as recipients in the bm12-->C57B6 cGVH model. CD4KO B6 mice injected with allogeneic bm12 spleen cells (bm12-->CD4KO group) showed no evidence of cGVH disease. They made no detectable autoantibodies, including anti-chromatin, anti-dsDNA, anti-ssDNA, and rheumatoid factor. They survived at least 20 wks after induction of cGVH disease; and they did not develop nephritis, based on the absence of detectable levels of proteinuria and normal renal histology at the time of sacrifice. By contrast, CD8KO B6 mice (bm12-->CD8KO group) and normal B6 mice (bm12-->B6 group) injected with bm12 spleen cells generally showed similar levels of mortality, nephritis, and autoantibodies, although the autoantibody titers declined somewhat after week 8 in the bm12-->CD8KO group. Control groups of recipients injected with B6 spleen cells showed no induction of autoantibodies. A surprising finding, however, was that the B6-->CD8KO group developed severe histologic glomerulonephritis in the absence of autoantibodies and with decreased immune deposits. These results indicate that endogenous (host) CD4+ T cells play an essential role in the cGVH autoimmune syndrome.     

Involvement of p53 and p21 in cellular defects and tumorigenesis in Atm-/- mice

Disruption of the mouse Atm gene, whose human counterpart is consistently mutated in ataxia-telangiectasia (A-T) patients, creates an A-T mouse model exhibiting most of the A-T-related systematic and cellular defects. While ATM plays a major role in signaling the p53 response to DNA strand break damage, Atm-/- p53(-/-) mice develop lymphomas earlier than Atm-/- or p53(-/-) mice, indicating that mutations in these two genes lead to synergy in tumorigenesis. The cell cycle G1/S checkpoint is abolished in Atm-/- p53(-/-) mouse embryonic fibroblasts (MEFs) following gamma-irradiation, suggesting that the partial G1 cell cycle arrest in Atm-/- cells following gamma-irradiation is due to the residual p53 response in these cells. In addition, the Atm-/- p21(-/-) MEFs are more severely defective in their cell cycle G1 arrest following gamma-irradiation than Atm-/- and p21(-/-) MEFs. The Atm-/- MEFs exhibit multiple cellular proliferative defects in culture, and an increased constitutive level of p21 in these cells might account for these cellular proliferation defects. Consistent with this notion, Atm-/- p21(-/-) MEFs proliferate similarly to wild-type MEFs and exhibit no premature senescence. These cellular proliferative defects are also rescued in Atm-/- p53(-/-) MEFs and little p21 can be detected in these cells, indicating that the abnormal p21 protein level in Atm-/- cells is also p53 dependent and leads to the cellular proliferative defects in these cells. However, the p21 mRNA level in Atm-/- MEFs is lower than that in Atm+/+ MEFs, suggesting that the higher level of constitutive p21 protein in Atm-/- MEFs is likely due to increased stability of the p21 protein.     

In vitro generation of soluble suppressor factor in murine chronic graft versus host (GVH) disease

Veto cells are cells which suppress self directed cytotoxicity (CTX) regardless of the strain of origin of the cytotoxic effector cell. The mechanism by which veto cells inhibit self directed CTX is largely unknown. We have previously described the emergence of a veto cell in murine chronic graft versus host (CGVH) disease both in vivo and in vitro. Using an in vitro system in which veto cells are induced, we now report that a soluble factor is also induced which is capable of suppressing CTX. This cytotoxicity suppressing factor (CsF) is heat labile and has a molecular weight above 50 kD. The factor prevents the emergence of a cytotoxic effector cell. but is largely ineffective once the effector cell has functionally emerged. CsF is irreversible and is not restricted in its ability to inhibit anti-self CTX.     

Involvement of protein kinase C and of protein phosphatases 1 and/or 2A in p47 phox phosphorylation in formylmet-Leu-Phe stimulated neutrophils: studies with selective inhibitors RO 31-8220 and calyculin A

Previously employed non-selective protein kinase inhibitors yielded inconclusive results regarding involvement of protein kinase C (PKC) in phosphorylation of 47 kDa protein (p47 phox) in intact neutrophils stimulated with physiologic agonists of superoxide generation. In the present study, phosphorylation of p47 phox in formylMet-Leu-Phe (fMLP) stimulated neutrophils was potently inhibited in the presence of 0.3 microM RO 31-8220, a selective inhibitor of PKC. These results provide experimental evidence in support of the currently considered essential involvement of PKC in p47 phox phosphorylation in response to physiologic stimulation of neutrophil surface receptors. The fMLP-induced phosphorylation of p47 phox was enhanced and prolonged by calyculin A, a specific inhibitor of protein phosphatases of types 1 and 2A, and such enhanced phosphorylation was also effectively inhibited by RO 31-8220. Our results suggest that the extent and duration of p47 phox phosphorylation in intact fMLP-stimulated neutrophils is probably controlled by a balance between the activities of PKC, on the one hand, and of protein phosphatase(s) of type(s) 1 and/or 2A, on the other. Effects of RO 31-8220 and of calyculin A on the fMLP-induced p47 phox phosphorylation were paralleled by similar effects on superoxide release. Calyculin A and RO 31-8220 were also used to study signal transduction by a post-receptor agonist of superoxide generation, a calcium ionophore A23187. The results of the latter study indicated that PKC was activated in A23187-stimulated neutrophils and was essentially involved in superoxide generation and p47 phox phosphorylation. Further, these results suggested that protein phosphatase(s) of type(s) 1 and/or 2A were also activated in A23187-signalling pathway, and limited the extent of superoxide release and p47 phox phosphorylation.     

Successful interferon-gamma therapy in a chronic granulomatous disease (CGD) patient suffering from Staphylococcus aureus hepatic abscess and invasive Candida albicans infection

Single photon emission computed tomography (SPECT) is being increasingly used as an adjunctive technique in the localisation of epileptogenic foci prior to surgery. Fourteen patients (five male, nine female; mean age 31 years) with refractory complex partial seizures, all of whom had abnormal 99Tc HM-PAO SPECT scans, were imaged again using the benzodiazepine receptor ligand 123I-iomazenil. Eleven of these patients displayed the same abnormality on both scans. The magnitude of the deficit, however, was slightly greater (P < 0.05) on the iomazenil scan compared with the HM-PAO study. These 11 patients were then randomised into two groups. Group 1 (n = 7) received clobazam 20 mg/day for a minimum of 7 days before a second iomazenil study was performed. Patients in group 2 (n = 4) underwent a second iomazenil scan at the same time interval as those in group 1, but without the additional clobazam. The resultant scans were reported blind to treatment. There were no qualitative or quantitative differences between the first and second iomazenil studies in either group. Patients can be imaged using 123I-iomazenil without withdrawing clobazam therapy. From this preliminary study, there seems little advantage in using iomazenil in place of HM-PAO to delineate the extent of the zone of epileptogenesis. Whether iomazenil SPECT will prove to be more sensitive in identifying the site of the epileptic focus remains to be determined.     

Bacillus thuringiensis subsp. konkukian (serotype H34) superinfection: case report and experimental evidence of pathogenicity in immunosuppressed mice

We present a case of severe war wounds infected by Bacillus thuringiensis serotype H34 and describe the experimental protocol used to demonstrate its ability to infect mice after cutaneous inoculation. This case is interesting because B. thuringiensis is considered to be a contaminant in laboratories and receives inadequate attention.     

Of mice (Mus musculus) and toddlers (Homo sapiens): Evidence for species-general spatial reorientation.

There is ongoing debate in spatial cognition about the mechanisms by which organisms are able to reorient, or reestablish a position, in the world after losing their bearing. The traditional view is that there is an encapsulated reorientation module that can only process environmental geometry such as distances or angles (Cheng, 1986). Recently, this view has been challenged on the grounds that the reorientation mechanism is only able to accept geometric information and may instead depend on a more general ability to use relative cues. J. Huttenlocher and S. F. Lourenco (2007) demonstrated that toddlers are successfully able to reorient with continuous cues but show remarkable deficits using categorical cues that are similar in perceptual complexity for reorientation. Here, the authors show the same pattern of results with mice (Mus musculus). These findings provide evidence that there is a homologous reorientation mechanism between mice and humans. Thus, future researchers can examine the genetic basis of this important cognitive ability. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Antiretroviral efficacy and pharmacokinetics of oral bis(isopropyloxycarbonyloxymethyl)-9-(2-phosphonylmethoxypropyl)adenine in mice

To overcome the low oral bioavailability of the highly potent and selective antiretroviral agent (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA), a new lipophilic ester derivative, i.e., the bis(isopropyloxycarbonyloxymethyl)-ester [bis(POC)-PMPA], was prepared. The usefulness of bis(POC)-PMPA as an oral prodrug for PMPA was investigated in the intestinal mucosa Caco-2 cell monolayer model. The total transport of bis(POC)-PMPA was 2.7%, whereas it was less than 0.1% for PMPA. Bis(POC)-PMPA was considerably metabolized inside the epithelial cells, since the majority of the compound was recovered after transport in the form of the monoester metabolite [mono(POC)-PMPA]. In contrast, bis(POC)-PMPA was relatively resistant to degradation at the luminal side of the Caco-2 cells. Pharmacokinetic studies with mice showed that the oral bioavailability of bis(POC)-PMPA (calculated from the curves of the concentration of free PMPA in plasma) was 20%. Neither bis(POC)-PMPA nor mono(POC)-PMPA could be recovered in plasma, suggesting the efficient release of the active drug PMPA after oral administration of bis(POC)-PMPA. Severe combined immunodeficient (SCID) mice infected with Moloney murine sarcoma virus (MSV) and treated orally with bis(POC)-PMPA for 5 or 10 days (dosages, 50, 100, or 200 mg of PMPA equivalent per kg of body weight per day) showed a significant delay in MSV-induced tumor appearance and tumor-associated death. The antiviral efficacy of oral bis(POC)-PMPA was related to the dosage and treatment period and was not significantly different from that of subcutaneous PMPA given at an equivalent dose. The favorable pharmacokinetic profile, marked antiviral efficacy, and low toxicity make bis(POC)-PMPA an attractive oral prodrug of PMPA that should be further pursued in clinical studies with patients infected with human immunodeficiency virus or hepatitis B virus.     

Bubble ring play of bottlenose dolphins (Tursiops truncatus): Implications for cognition.

Research on the cognitive capacities of dolphins and other cetaceans (whales and porpoises) has importance for the study of comparative cognition, particularly with other large-brained social mammals, such as primates. One of the areas in which cetaceans can be compared with primates is that of object manipulation and physical causality, for which there is an abundant body of literature in primates. The authors supplemented qualitative observations with statistical methods to examine playful bouts of underwater bubble ring production and manipulation in 4 juvenile male captive bottlenose dolphins (Tursiops truncatus). The results are consistent with the hypothesis that dolphins monitor the quality of their bubble rings and anticipate their actions during bubble ring play. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Differences between chimpanzees (Pan troglodytes) and humans (Homo sapiens) in the resting state of the index finger: Implications for pointing.

Pointing with the index finger is a universal human (Homo sapiens) behavior found in cultures around the world. The emergence of pointing in infancy has traditionally been linked with the early development of cognitive capacities such as perspective-taking, intersubjectivity, and empathy. In contrast to humans, chimpanzees (Pan troglodytes) do not develop a pointing gesture with the index finger and rarely point by gesturing with hands or arms. However, recent experimental data suggest that chimpanzees may develop cognitive skills related to perspective-taking and empathy. In this article we report the existence of a difference in the resting morphology of the index finger in humans and chimpanzees. We speculate on how this species difference may be related to species differences in pointing with the index finger. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

On the key role of secondary lymphoid organs in antiviral immune responses studied in alymphoplastic (aly/aly) and spleenless (Hox11(-)/-) mutant mice

The role of the spleen and of other organized secondary lymphoid organs for the induction of protective antiviral immune responses was evaluated in orphan homeobox gene 11 knockout mice (Hox11(-/-)) lacking the spleen, and in homozygous alymphoplastic mutant mice (aly/aly) possessing a structurally altered spleen but lacking lymph nodes and Peyer's patches. Absence of the spleen had no major effects on the immune response, other than delaying the antibody response by 1-2 d. In aly/aly mice, the thymus-independent IgM response against vesicular stomatitis virus (VSV) was delayed and reduced, whereas the T-dependent switch to the protective IgG was absent. Therefore, aly/aly mice were highly susceptible to VSV infection. Since aly/aly spleen cells yielded neutralizing IgM and IgG after adoptive transfer into recipients with normally structured secondary lymphoid organs, these data suggest that the structural defect was mainly responsible for inefficient T-B cooperation. Although aly/aly mice generated detectable, but reduced, CTL responses after infection with vaccinia virus (VV) and lymphocytic choriomeningitis virus (LCMV), the elimination of these viruses was either delayed (VV) or virtually impossible (LCMV); irrespective of the dose or the route of infection, aly/aly mice developed life-long LCMV persistence. These results document the critical role of organized secondary lymphoid organs in the induction of naive T and B cells. These structures also provide the basis for cooperative interactions between antigen-presenting cells, T cells, and B cells, which are a prerequisite for recovery from primary virus infections via skin or via blood.     

BBB transport and P-glycoprotein functionality using MDR1A (-/-) and wild-type mice. Total brain versus microdialysis concentration profiles of rhodamine-123

PURPOSE: The effect of P-glycoprotein (Pgp) on brain distribution using mdr1a (-/-) mice was investigated. METHODS: Fluorescein (Flu) and FD-4 were used to check whether blood-brain barrier (BBB) integrity was maintained in mdr1a (-/-) mice. The Pgp substrate rhodamine-123 (R123) was infused and total brain, blood and brain microdialysate concentrations in mdr1a (-/-) mice and wild-type mice were compared. RESULTS: Maintenance of BBB integrity was indicated by equal total brain/blood ratios of Flu and FD-4 in both mice types. R123 concentrations in brain after i.v. infusion were about 4-fold higher in mdr1a (-/-) than in wild-type mice (P < 0.05), without changes in blood levels. After microdialysis experiments the same results were found, excluding artifacts in the interpretation of Pgp functionality by the use of this technique. However the 4-fold ratio in brain was not reflected in corresponding microdialysates. No local differences of R123 in the brain were found. By the no-net-flux method in vivo recovery appeared to 4.6-fold lower in mdrla (-/-) mice compared with wild-type mice. CONCLUSIONS: Pgp plays an important role in R123 distribution into the brain. Using intracerebral microdialysis, changes in in vivo recovery by the absence or inhibition of Pgp (or active efflux in general) need to be considered carefully.