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Osteocytes sense the microenvironmental stimuli, including mechanical stress, and regulate bone resorption by osteoclasts and bone formation by osteoblasts. Diabetes and cancer metastasis to bone raise l-lactic acid in the bone tissue, causing acidification. Here, we investigated the effects of l-lactic acid and extracellular acidification on the function of mouse Ocy454 osteocytes. L- and d-lactic acid with low chiral selectivity and acidification of the medium raised the production of sclerostin and osteoprotegerin by Ocy454 cells. The mRNA expression of their genes increased after either treatment of L- and d-lactic acid or acidification of the medium. Furthermore, the conditioned medium of Ocy454 cells cultured in an acidic environment suppressed the induction of alkaline phosphatase activity in MC3T3-E1 cells, which was recovered by the anti-sclerostin antibody. While it is reported that HDAC5 inhibits the transcription of the sclerostin gene, extracellular acidification reduced the nuclear localization of HDAC5 in Ocy454 cells. While calmodulin kinase II (CaMKII) is known to phosphorylate and induce extranuclear translocation of HDAC5, KN-62, an inhibitor of CaMKII lowered the expression of the sclerostin gene in Ocy454 cells. Collectively, extracellular acidification is a microenvironmental factor that modulates osteocyte functions.

Osteoclasts are multinucleated giant cells differentiated from monocyte-macrophage-lineage cells under stimulation of receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) produced by osteoblasts and osteocytes. Although it has been reported that nitric oxide (NO) and reactive oxygen species (ROS) are involved in this process, the mechanism by which these labile molecules promote osteoclast differentiation are not fully understood. In this study, we investigated the formation and function of 8-nitro-cGMP, a downstream molecule of NO and ROS, in the process of osteoclast differentiation in vitro. 8-Nitro-cGMP was detected in mouse bone marrow macrophages and osteoclasts differentiated from macrophages in the presence of RANKL. Inhibition of NO synthase suppressed the formation of 8-nitro-cGMP as well as RANKL-induced osteoclast differentiation from macrophages. On the other hand, RANKL-induced osteoclast differentiation was promoted by addition of 8-nitro-cGMP to the cultures. In addition, 8-nitro-cGMP enhanced the mRNA expression of RANK, the receptor for RANKL. However, 8-bromo-cGMP, a membrane-permeable derivative of cGMP, did not have an effect on either RANKL-induced osteoclast differentiation or expression of the RANK gene. These results suggest that 8-nitro-cGMP is a novel positive regulator of osteoclast differentiation, which might help to explain the roles of NO and ROS in osteoclast differentiation.

Objectives Intraoperative identification of tumor margins is essential to achieving complete tumor resection. However, the process of intraoperative pathological diagnosis involves cumbersome procedures, such as preparation of cryosections and microscopic examination, thus requiring more than 30 min. Moreover, intraoperative diagnoses made by examining cryosections are occasionally inconsistent with postoperative diagnoses made by examining paraffin-embedded sections because the former are of poorer quality. We sought to establish a more rapid accurate method of intraoperative assessment. Materials and methods A diagnostic algorithm of head and neck squamous cell carcinoma (HNSCC) using machine learning was constructed by mass spectra obtained from 15 non-cancerous and 19 HNSCC specimens by probe electrospray ionization mass spectrometry (PESI-MS). The clinical validity of this system was evaluated using intraoperative specimens of HNSCC and normal mucosa. Results A total of 114 and 141 mass spectra were acquired from non-cancerous and cancerous specimens, respectively, using both positive- and negative-ion modes of PESI-MS. These data were fed into partial least squares-logistic regression (PLS-LR) to discriminate tumor-specific spectral patterns. Leave-one-patient-out cross validation of this algorithm in positive- and negative-ion modes showed accuracies in HNSCC diagnosis of 90.48% and 95.35%, respectively. In intraoperative specimens of HNSCC, this algorithm precisely defined the borders of the cancerous regions; these corresponded with those determined by examining histologic sections. The procedure took approximately 5 min. Conclusion This diagnostic system, based on machine learning, enables accurate discrimination of cancerous regions and has the potential to provide rapid intraoperative assessment of HNSCC margins.

Melanin-concentrating hormone (MCH) receptor 1 (MCHR1) is a class A G-protein-coupled receptor (GPCR). The MCH-MCHR1 system has been implicated in the regulation of feeding, emotional processing, and sleep in rodents. Recent work revealed that MCHR1 is selectively expressed in neuronal primary cilia of the central nervous system. Cilia have various chemosensory functions in many types of cell, and ciliary dysfunction is associated with ciliopathies such as polycystic kidney disease and obesity. Although dynamic modulation of neuronal cilia length is observed in obese mice, the functional interaction of neuronal ciliary GPCR and its endogenous ligand has not yet been elucidated. We report here that MCH treatment significantly reduced cilia length in hTERT-RPE1 cells (hRPE1 cells) transfected with MCHR1. Quantitative analyses indicated that MCH-induced cilia shortening progressed in a dose-dependent manner with an EC50 lower than 1nM when cells were treated for 6h. Although the assembly and disassembly of primary cilia are tightly coupled to the cell cycle, cell cycle reentry was not a determinant of MCH-induced cilia shortening. We confirmed that MCH elicited receptor internalization, Ca(2+) mobilization, ERK and Akt phosphorylation, and inhibition of cyclic AMP accumulation in MCHR1-expressing hRPE1 cells. Among these diverse pathways, we revealed that Gi/o-dependent Akt phosphorylation was an important component in the initial stage of MCH-induced cilia length shortening. Furthermore, induction of fewer cilia by Kif3A siRNA treatment significantly decreased the MCH-mediated phosphorylation of Akt, indicating the functional importance of the MCHR1-Akt pathway in primary cilia. Taken together, the present data suggest that the MCH-MCHR1 axis may modulate the sensitivity of cells to external environments by controlling the cilia length. Therefore, further characterization of MCHR1 as a ciliary GPCR will provide a potential molecular mechanism to link cilia length control with obesity.

Background Degeneration of articular cartilage including reduced cellularity is often observed in the pathogenesis of osteoarthritis. We investigated nitric oxide (NO·)- and reactive oxygen species (ROS)-dependent cell death induced by interleukin-1β (IL-1β) in mouse chondrocyte-like ATDC5 cells and rat primary chondrocytes in vitro. Increased production of lactate was observed in IL-1β-treated ATDC5 cells before beginning of their death. Cell death was suppressed by introducing small interfering RNA (siRNA) for monocarboxylate transporter-1 (MCT-1), a membrane transporter for lactate and pyruvate distributed in plasma and mitochondrial inner membranes. Highlight MCT-1 gene silencing prevented IL-1β-induced expression of phagocyte-type NADPH-oxidase (NOX-2), an enzyme specialized for ROS production; there was no effect on the expression of inducible NO· synthase (iNOS). IL-1β-induced cell death was suppressed by NOX-2 siRNA, indicating involvement of this enzyme in cell death. Although phosphorylation and degradation of inhibitor of κBα (I-κBα) from 5 to 20min after addition of IL-1β was not affected by MCT-1 siRNA, degradation of I-κBα and nuclear translocation of RelA/p65 observed in control cells from 36 to 48h after exposure to IL-1β was not seen in MCT-1-silenced cells. Scavenging of ROS suppressed both late-phase I-κBα degradation and NOX-2 expression. MCT-1 siRNA lowered the level of ROS generated after 15h exposure to IL-1β. Conclusion We found that MCT-1 contributed to the expression of NOX-2 via late-phase activation of nuclear factor κB in a ROS-dependent manner in cells exposed to IL-1β. Hence, MCT-1 could be a potential target for the treatment of degenerative joint diseases.

It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and β-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and β-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05-5 mmol/L). In contrast, β-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by β-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or β-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations.

Neutrophils are one of the most abundant leukocytes in the sites of lesion of inflammatory diseases such as periodontitis and rheumatoid arthritis. These diseases are accompanied by bone loss, which worsens the quality of life of the patients. However, the role of neutrophils in the inflammatory bone loss has not been fully investigated. In the present study, we found that human neutrophils enhanced osteoclast differentiation from mouse bone marrow cells co-cultured with mouse osteoblasts in the presence of active vitamin D3. The enhanced osteoclast differentiation was significantly suppressed by elastatinal, a synthetic inhibitor of neutrophil elastase. Also, we found that human neutrophils degraded human recombinant osteoprotegerin (OPG), a decoy receptor for nuclear factor κB (RANK) ligand (RANKL), the essential osteoclast differentiation-inducing factor, expressed by osteoblasts. Degradation of OPG by neutrophils was suppressed by human α1-protease inhibitor, the major endogenous inhibitor of neutrophil elastase. Recombinant human neutrophil elastase degraded human OPG in its death domain-like region. These results indicated that the degradation of OPG by elastase contributed at least in part to the enhanced osteoclast differentiation by neutrophils. There is a possibility that neutrophils play an important role in inflammatory bone loss.

Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159–166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1β in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1β were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.

Real-time analyses of hepatocellular carcinoma were performed in living mice to assess the applicability of probe electrospray ionization-mass spectrometry (PESI-MS) in medical diagnosis. The number of peaks and the abundance of ions corresponding to triacylglycerols (TAGs) were higher in cancerous tissues than in noncancerous tissues. Multiple sequential scans of the specimens were performed along a predetermined line extending over the noncancerous region to detect the boundary of the cancerous region. Our system successfully discriminated the noncancerous and cancerous tissues based on the intensities of the TAG ions. These results highlight the potential application of PESI-MS for clinical diagnosis in cancer.

Myelination is an essential prerequisite for the nervous system to transmit an impulse efficiently by a saltatory conduction. In the peripheral nervous system (PNS), Schwann cells (SCs) engage in myelination. However, a detailed molecular mechanism underlying myelination still remains unclear. In this study, we hypothesized that the primary cilia of SCs are the regulators of Hedgehog (Hh) signaling-mediated myelination. To confirm our hypothesis, we used mouse dorsal root ganglion (DRG)/SC co-cultures, wherein the behavior of SCs could be analyzed by maintaining the interaction of SCs with DRG neurons. Under these conditions, SCs had primary cilia, and Hh signaling molecules accumulated on the primary cilia. When the SCs were stimulated by the addition of desert hedgehog or smoothened agonist, formation of myelin segments on the DRG axons was facilitated. On the contrary, upon administration of cyclopamine, an inhibitor of Hh signaling, myelin segments became comparable to those of controls. Of note, the ratio of SCs harboring primary cilium reached the highest point during the early phase of myelination. Furthermore, the strongest effects of Hh on myelination were encountered during the same stage. These results collectively indicate that Hh signaling regulates myelin formation through primary cilia in the PNS.

Probe electrospray ionization (PESI) is one of the most promising methods in biochemical analysis because it enables us to analyze biological samples very quickly without any special pretreatment. Moreover, due to the small size of the needle tip, this method has advantages such as low invasiveness to the samples, making it possible to analyze the biological profiles of organs or tissues in living animal in situ. In this study, we performed a real-time analysis of living mice that delineates the differences in lipid composition of hepatocytes between normal and steatotic mice. In steatotic mice, the number of peaks and the ion abundance for triacylglycerols were much higher compared with those of control mice. All mice used in this study tolerated the procedure well and survived for more than a month until sacrificed for further analysis. To test a potential for medical diagnosis, human tumor tissues were also measured and we obtained discriminative results judged as useful for diagnostics. These results pave the way into the application of PESI to the in vivo analysis of biological molecules.

POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.

Klotho mutant mice, defective in the klotho gene, develop multiple age-related disorders with very short lifespans. Introduction of the exogenous klotho gene into these mutant mice leads to an improvement in their phenotypes, while overexpression of this gene in wild-type mice significantly extends their lifespan. These observations suggest that the klotho gene/protein has an anti-aging function. Since there have been only a few reports with some disagreement about results on the CNS of the mutant mice, we tried to clarify whether the CNS neurons generate aging-like features, even in premature stages, using biochemical and morphological approaches. Results obtained from the mutant mice, when compared with wild-type mice, were as follows. Neurofilaments (NFs) were increased significantly in axons, with the subunit proteins showing a significant enhancement in phosphorylation or expression of NF-H or NF-L, respectively. Microtubules in Purkinje cell dendrites were closer to each other, and in the CNS tissue tubulin was unaltered, but microtubule-associated protein (MAP) 2 was significantly reduced in expression. Neuronal cellular organelles were morphologically disordered. Lysosomes, cathepsin D and light chain 3 of MAP1A/B (LC3) were augmented with the appearance of putative autophagy-related structures. Antiapoptotic Bcl-xL and proapoptotic Bax were reduced and enhanced, respectively, and mitogen-activated protein kinase was reduced. Synapse-related proteins and structures were decreased. Neuronal degeneration was evident in hippocampal pyramidal cells, and possibly in Purkinje cells. Astrocytic glial filaments and glial fibrillary acidic protein were increased in density and expression, respectively. Together, the CNS neuronal alterations in klotho mutant mice were quite similar to those found in aged animals, including even premature death, so this mouse should be a more appropriate animal model for CNS aging than those previously reported.

The Multi Pixel Photon Counter is a novel semiconductor photon sensor which consists of 100–1600 micro APD pixels. Each pixel works in limited Geiger mode with an inverse bias voltage around 80 V. The MPPC has many remarkable features such as high gain, large photon detection efficiency, low cost and tolerance for magnetic field. Fundamental properties, such as the gain, dark noise rate and inter-pixel cross-talk, have been measured with three different samples which have 100, 400, and 1600 pixels. Variation of the gain over 750 samples, and uniformity of the gain and photon sensitivity within one pixel are also evaluated. As the result of the tests, it is confirmed that the MPPC has satisfactory performance for many applications, not only for high energy physics experiments but also for medical engineering systems.

Recent economic developments in Japan suggest that there is a growing interest in how monetary policy should be conducted under low inflation and nominal interest rates. In this paper, we first confirm the effectiveness of conventional wisdom against a liquidity trap, namely taking aggressive responses around the zero bound and variant of price level targeting so that it can intensify the policy duration effect, in the large scale dynamic general equilibrium model, the JEM (Japanese Economic Model). We then show that the optimal tangible policy rules around the zero bound are possibly state-contingent at the same time. J. Japanese Int. Economies 20 (3) (2006) 364–379.

Nephronectin (Npnt) is an extracellular matrix protein known to be a ligand for the integrin α8β1. We previously demonstrated that Npnt expression was suppressed by TGF-β through ERK1/2 and JNK in osteoblasts. In this study, we found that inhibition of a TGF-β type I receptor (TGF-β R1, Alk5) by a specific inhibitor {2-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine} strongly induced Npnt expression in osteoblast-like MC3T3-E1 cells. The Alk5 inhibitor-induced increase of Npnt expression occurred in both time- and dose-dependent manners, while that expression was also induced by introduction of an siRNA for Smad2, a central intracellular mediator of TGF-β signaling. These results suggest that the expression of Npnt is regulated by the Alk5-SMAD signaling pathway in osteoblasts.

Resistance to Angiostrongylus cantonensis is contingent upon the generation of an eosinophilic response in the CSF of infected hosts. We have studied the parameters required for the generation of this CSF eosinophilia in normally permissive rats. We initially induced a marked peripheral eosinophilia in rats by infection with either Mesocestoides corti or Angiostrongylus cantonensis or the surgical transfer of A. cantonensis young adult worms (YA) into their pulmonary arteries. Next, we injected various antigens into the ventricles of these rats. A. cantonensis-preinfected rats demonstrated significant CSF eosinophilia following injection of A. cantonensis egg antigen, 1st-stage larval (L1) antigen, or M. corti antigen, but not following YA antigen inoculation. A. cantonensis egg and M. corti antigens were potent chemoattractants for eosinophils in an in vitro chemotaxis assay. These data indicate that peripheral eosinophilia, meningeal stimulation by A. cantonensis infection and the presence of potent chemoattractants, e.g., egg and L1 antigens are prerequisites for CSF eosinophil accumulation in permissive rat hosts.

Male BALB/c mice were infected with Angiostrongylus cantonensis and at various times p.i. treated with cyclosporin A (CsA) either for 5 days continuously or intermittently, or for 12-16 days on alternate days. They were monitored for peripheral blood eosinophilia and at necropsy examined for CSF and bone marrow eosinophilia, and worm recovery. CsA treatment provoked a transient inhibition of peripheral blood eosinophilia in all groups examined, followed by rebounding eosinophilia. There was no significant difference in bone marrow and CSF eosinophilia between CsA- and vehicle-treated groups. Mice treated with CsA on alternate days yielded lower intracranial worm recovery with small-sized worms at days 7, 21 and 30 p.i. than vehicle-treated groups did. No significant reduction in worm recovery was noted in mice treated for 5 days either continuously or intermittently, although worms, especially female ones, harvested from groups treated with CsA from days--1 to 3 or from days 13 to 17 were significantly smaller than those from vehicle-treated groups. CsA-treatment suppressed blastogenic responses of spleen cells against Con A or worm antigen at days 7 and 21 p.i. In vitro treatment of the 3rd stage larvae with CsA did not adversely influence survival of A. cantonensis in mice. These data indicate that CsA exerts anti-parasitic effects against A. cantonensis in mice.

The influence of Mesocestoides corti on subsequent Angiostrongylus cantonensis infection in mice (C57BL/6 and BALB/c) was assessed. Both strains of mice infected with M. corti demonstrated a peak blood eosinophilia at around 3 weeks post-infection (p.i.). C57BL/6 and BALB/c mice primarily infected with M. corti were given A. cantonensis infection 18 days later, but pre-existing M. corti infection did not affect the recovery of intracranial worms of A. cantonensis at day 21 p.i. BALB/c mice with mixed parasite infections showed low morbidity and mortality as compared with mice singly infected with A. cantonensis and some mice demonstrated a pulmonary migration of intracranial worms. In C57BL/6 mice, intracranial worms were killed and thus all mice survived. C57BL/6 mice with mixed parasite infections failed to resist A. cantonensis reinfection. The blastogenic responses of spleen cells against A. cantonensis antigen were lower in BALB/c than in C57BL/6 mice and mixed parasite infections also resulted in less blastogenic responses against both concanavalin A and A. cantonensis antigen than monoinfection. The recovery of M. corti biomass was significantly higher in mice with mixed parasite infections than mice with monoinfection with M. corti. These data suggest a distinct difference in response to A. cantonensis infection between C57BL/6 and BALB/c mice, and the induction of immunosuppression in both mouse strains following M. corti infection. Blood eosinophilia provoked by M. corti infection is not directly associated with the killing of worms in subsequent A. cantonensis infection.

Angiostrongylus cantonensis is the causative agent of human eostnophilic meningoencephalitis in the Pacific Islands and Southeast Asia. Prominent eosinophilia in the cerebrospinal fluid (CSF) of the patients has been used as one of the diagnostic criteria for the disease but the role(s) of the CSF eosinophils has remained to be elucidated. In this article, Kentaro Yoshimura, Hiroko Sugaya and Kazuto Ishido discuss the involvement of CSF eosinophils in the killing of introcranial worms and the damage of the central nervous system of the hosts, and consider why eosinophils in A. cantonensis infection play a more important role in nonpermissive hosts (including humans) than in the permissive rat host.

The kinetics of eosinophil growth and/or survival stimulating factor (Eo-stimulating factor) production in Angiostrongylus cantonensis-infected rats was assessed by in vitro marrow cultures. When lymphocytes, obtained from cervical, mediastinal or mesenteric lymph nodes of infected WKAH rats, were cultured with A. cantonensis antigens, Eo-stimulating factor activity was detected in the conditioned media obtained only from mediastinal lymph node cells at and after 35 days p.i. Eo-stimulating factor activity in the conditioned media was not detected before or at 20 days p.i. The kinetics of the factor was, therefore, almost identical to those of eosinophilia in bone marrow and peripheral blood. This Eo-stimulating factor activity was inhibited (maximum inhibition = 73%) by anti-mouse IL-5 monoclonal antibody in a dose-dependent fashion, indicating that the factor would be, at least in part, identical to IL-5. Similar Eo-stimulating factors were also detected in the conditioned media which were obtained by stimulating spleen cells from Wistar rats at 35–45 days p.i. with Con A. These data suggest that lymphocytes in the mediastinal lymph nodes, local lymph nodes near the adult worm habitat, could play an essential role in induction of eosinophilia in A. cantonensis-infected rats.

The survival, growth, and egg-laying capacity of young adult Angiostrongylus cantonensis, surgically transferred from intracranial sites into pulmonary arteries, were studied. A variety of experimental animals (rats, guinea pigs, mice, and mastomys) were chosen as donor animals and as recipient hosts (rats, guinea pigs, and rabbits). These species were specifically chosen to span the spectrum of host permissiveness relative to worm development in an attempt to understand the mechanisms which underlie species-dependent resistance. Recipient animals were monitored not only for the development of parasites per se but also for antibody production and histopathologic changes. The results indicated that these procedures were technically feasible, with good worm development following intra-rat transfers, as early as 15 days after initial exposure. Studies were performed to analyze the constraints of development both on initial, i.e., prelung and subsequent i.e., postlung development. When worms were obtained from permissive species such as rat or mastomys, transfer into rats resulted in good growth and development; however, worms which developed initially in exposed mice or guinea pigs developed less well in the rat. Conversely, worms which developed initially in permissive host such as the rat, when transferred into a variety of less permissive hosts such as the guinea pig and rabbit, apparently did not survive and caused significant morbidity and mortality within the nonpermissive host. Histopathologic evaluation revealed a strong eosinophilic perivascular and peribronchiolar infiltration as well as granulomatous reactions surrounding the worms in the lungs of recipient guinea pigs and rabbits, changes not observed in the lungs of permissive rat recipients. As reaginic antibody responses were also more prominent in nonpermissive than in permissive animals, it is possible that IgE responses may be more directly related to the phenomenon of morbidity and/or permissiveness than are other aspects of immune response. In support of this contention was the finding of nearly equivalent hemagglutinating antibody production between permissive rats and nonpermissive guinea pigs and rabbits.

Specific characteristics of Paragonimus westermani, P. ohirai , and P. miyazakii were sought in electrophoretic patterns of whole body proteins. From P. westermani , a minimum of 23 distinct protein bands was separated. Densitometric tracings gave at least 6 prominent peaks considered to be species specific. A minimum of 24 distinct bands was identified from P. ohirai . At least 7 of the bands were prominent and considered characteristic of P. ohirai . No significant difference was found in the electrophoretic patterns and the R F value of each band separated from P. ohirai worms obtained from rats and cats. A minimum of 23 distinct bands was identified from P. miyazakii . This species has at least 6 prominent bands, of which two (bands 7 and 11) were especially high in density. There was observed no significant difference between P. miyazakii worms originating from crabs collected at Iwakuni or at Kurodasho. The three species of lung flukes showed similar electrophoretic patterns in the gel regions near the origin and the front band. Two prominent bands identified from these gel regions were common to all of the above species. The major differences are found in the mid region of the gel. Each of the three lung flukes has a species-characteristic electrophoretic pattern containing several prominent protein fractions. These patterns are highly reproducible and definitive for all of the species.