[A Case of Anti-Resorptive Agents-Related Osteonecrosis of the Jaw and Extensive Loss of Maxillary Bone in a Patient of Breast Cancer].

A 74-year-old woman had a mastectomy for right breast cancer in 201X. Eight years later, the patient developed multiple bone metastases and was treated with denosumab by her previous doctor. A year after, she was diagnosed with anti-resorptive agents-related osteonecrosis of the jaw, and preservation treatment was performed. In 201X+11, the patient had difficulty walking and was admitted to our palliative care ward. A month later, her maxillary bone detached extensively and spontaneously. By applying infection preventive measures and in cooperation with the former doctor, dentist, and oral surgeon, as well as visiting the dental department in our hospital, the patient managed to continue eating what she liked. Jaw infection did not occur. The patient died of liver dysfunction due to liver metastasis 5 months following the extensive loss of the maxillary bone.

The Lack of Bmal1, a Core Clock Gene, in the Intestine Decreases Glucose Absorption in Mice.

The circadian clock network is an evolutionarily conserved system that regulates systemic metabolism, such as glucose homeostasis. Intestinal tissue is a pivotal organ for the regulation of glucose metabolism, mainly via glucose absorption into the circulation; however, the significance of the intestinal circadian clock network for glucose metabolism remains largely unclear. We herein utilized a mouse model in which Bmal1, a core clock gene, was deleted in an intestine-specific manner (Bmal1Int-/- mice) and demonstrated a rhythmic expression of Sglt1 with its peak at zeitgeber time (ZT) 10.7 ±â€…2.8 in control mice, whereas this was lost in Bmal1Int-/- mice. Mechanistically, chromatin immunoprecipitation analysis revealed rhythmic binding of CLOCK to the E-box elements in the Sglt1 gene in control mice; however, this was absent in Bmal1Int-/- mice. Accordingly, SGLT1 protein levels were decreased during the dark phase in Bmal1Int-/- mice and this was associated with impaired glucose absorption, leading to a decline in hepatic glycogen levels at ZT4, which was restored by ingestion of high-sucrose water. Additionally, when mice were starved from ZT0, greater expression of the lipolysis-related gene Pnpla2 was observed in adipose tissue of Bmal1Int-/- mice, and this was not noted when glycogen storage was restored by high-sucrose water prior to fasting, suggesting that higher Pnpla2 expression in Bmal1Int-/- mice was likely caused by lower glycogen storage. These results indicate that disruption of the intestinal circadian clock system impairs glucose absorption in the intestine and affects systemic glucose homeostasis.

Clonal osteoblastic cell lines with CRISPR/Cas9-mediated ablation of Pit1 or Pit2 show enhanced mineralization despite reduced osteogenic gene expression.

Multiple actions of extracellular Pi on the skeletal cells are likely to be partly mediated by type III sodium/phosphate (Na+/Pi) cotransporters Pit1 and Pit2, although the details are not fully understood. In the current study, to determine the roles of Pit1 and Pit2 in osteoblasts, we generated Pit1-knockout (KO) and Pit2-KO osteoblastic cells by applying CRISPR/Cas9 genome editing to an osteoblastic cell line MC3T3-E1 subclone 4. The extracellular Pi level was increased in the Pit1-KO and Pit2-KO clones due to the reduced Pi uptake. Interestingly, in vitro mineralization was accelerated in the Pit1-KO and Pit2-KO clones, although the induction of the expression of osteogenic marker genes was suppressed. In the cells before mineralization, extracellular levels of pyrophosphate (PPi) and adenosine triphosphate (ATP) were increased in the Pit1-KO and Pit2-KO clones, which might be attributable to the reduced expression and activity of tissue-nonspecific alkaline phosphatase (TNSALP). A 24-h treatment with high Pi reduced the expression and activity of TNSALP, suggesting that the suppression of TNSALP in the Pit1-KO and Pit2-KO clones was caused by the increased availability of extracellular Pi. Lentiviral gene transfer of Pit1 and Pit2 restored the changes observed in Pit1-KO and Pit2-KO clones, respectively. The expressions of P2Y2 and P2X7 which encode receptors for extracellular ATP were altered in the Pit1-KO and Pit2-KO clones, suggesting an influence on purinergic signaling. In mineralized cells after long-term culture, intracellular levels of PPi and ATP were higher in the Pit1-KO and Pit2-KO clones. Taken together, ablation of Pit1 or Pit2 in this osteoblastic cell model led to accelerated mineralization, suppressed TNSALP and altered the levels of extracellular and intracellular PPi and ATP, which might be partly mediated by changes in the availability of extracellular Pi.

Lack of PTEN in osteocytes increases circulating phosphate concentrations by decreasing intact fibroblast growth factor 23 levels.

Fibroblast growth factor 23 (FGF23) has been centric to the regulation of phosphate (Pi) metabolism; however, the regulatory network of FGF23 in osteocytes has not yet been defined in detail. We herein investigated the role of PTEN (phosphatase and tensin homolog deleted from chromosome 10) in this regulation. We created mice lacking PTEN expression mainly in osteocytes by crossing Pten-flox mice with Dmp1-Cre mice. The lack of PTEN in the osteocytes of these mice was associated with decreased skeletal and serum intact FGF23 levels, which, in turn, resulted in reductions of urinary Pi excretion and elevations of serum Pi levels. Mechanistically, the knockdown of PTEN expression in osteoblastic UMR106 cells activated the AKT/mTORC1 (mechanistic target of rapamycin complex 1) pathway and this was associated with reductions in Fgf23 expression. Furthermore, the suppression of Fgf23 expression by PTEN knockdown or insulin simulation in UMR106 cells was partially restored by the treatment with the mTORC1 inhibitor, rapamycin. These results suggest that FGF23 expression in osteoblastic cells is in part regulated through the AKT/mTORC1 pathway and provide new insights into our understanding of the regulatory network of Pi metabolism.

Intestinal clock system regulates skeletal homeostasis.

The circadian clock network is an evolutionarily conserved system involved in the regulation of metabolic homeostasis; however, its impacts on skeletal metabolism remain largely unknown. We herein demonstrated that the circadian clock network in the intestines plays pivotal roles in skeletal metabolism such that the lack of the Bmal1 gene in the intestines (Bmal1Int-/- mice) caused bone loss, with bone resorption being activated and bone formation suppressed. Mechanistically, Clock protein interaction with the vitamin D receptor (VDR) accelerated its binding to the VDR response element by enhancing histone acetylation in a circadian-dependent manner, and this was lost in Bmal1Int-/- mice because nuclear translocation of Clock required the presence of Bmal1. Accordingly, the rhythmic expression of VDR target genes involved in transcellular calcium (Ca) absorption was created, and this was not observed in Bmal1Int-/- mice. As a result, transcellular Ca absorption was impaired and bone resorption was activated in Bmal1Int-/- mice. Additionally, sympathetic tone, the activation of which suppresses bone formation, was elevated through afferent vagal nerves in Bmal1Int-/- mice, the blockade of which partially recovered bone loss by increasing bone formation and suppressing bone resorption in Bmal1Int-/- mice. These results demonstrate that the intestinal circadian system regulates skeletal bone homeostasis.

Inorganic Phosphate Activates the AKT/mTORC1 Pathway and Shortens the Life Span of an α­Klotho-Deficient Model.

Inorganic phosphate (Pi) has been implicated in the pathogenesis of accelerated aging; however, the underlying mechanisms remain elusive. Herein, we demonstrated in cultured cells and in vivo that increased levels of extracellular Pi activated the AKT/mammalian target of rapamycin complex 1 (mTORC1) pathway by suppressing membrane-bound phosphatase and tensin homolog (PTEN) levels in a manner requiring the sodium-dependent Pi transporter PiT­1. High levels of extracellular Pi also led to phosphorylation of Ser/Thr clusters in the C­terminal tail of PTEN, which has been shown to dissociate PTEN from the membrane. Notably, blockade of mTORC1 activity by rapamycin treatment prolonged the life span of hyperphosphatemic α­Klotho-deficient (Kl(-/-)) mice. Dietary correction of hyperphosphatemia or treatment with rapamycin also rescued the brown adipose tissue dysfunction and oxidative damage observed in Kl(-/-) mice. Furthermore, rapamycin treatment partially rescued these effects and extended the life span when Kl(-/-) mice were maintained on a high-phosphate diet. Finally, rapamycin reduced circulating Pi levels in Kl(-/-) mice, apparently by decreasing the localization of sodium-dependent Pi transport protein 2a at the renal brush border membrane. Therefore, the activation of mTORC1 may create a vicious loop that exacerbates the retention of Pi, which in turn may enhance oxidative damage and ultimately shorten the life span of Kl(-/-) mice. These results demonstrate that Pi has important roles in the aging process, and the blockade of mTORC1 may have therapeutic potential for premature aging-like symptoms associated with hyperphosphatemia.

Effect of dietary fish oil on mouse testosterone level and the distribution of eicosapentaenoic acid-containing phosphatidylcholine in testicular interstitium.

Low levels of serum testosterone are characteristically associated with diabetes, coronary atherosclerosis, obstructive sleep apnea, rheumatoid arthritis, and chronic obstructive pulmonary disease. Testosterone replacement therapy is effective against many of these disorders, indicating the importance of maintaining a healthy testosterone level. In this study, we investigated the effects of fish oil on murine testosterone metabolism and analyzed the dynamics of relevant lipids in testes by matrix-assisted laser desorption ionization mass spectrometry imaging. Testosterone was upregulated in mice that received fish oil. In the testicular interstitium, eicosapentaenoic acid-containing phosphatidylcholine was distributed characteristically. These data suggest that eicosapentaenoic acid is involved in testosterone metabolism.

Class switch recombination and somatic hypermutation of virus-neutralizing antibodies are not essential for control of friend retrovirus infection.

Toll-like receptor 7 and Myd88 are required for antiretroviral antibody and germinal center responses, but whether somatic hypermutation and class-switch recombination are required for antiretroviral immunity has not been examined. Mice deficient in activation-induced cytidine deaminase (AID) resisted Friend virus infection, produced virus-neutralizing antibodies, and controlled viremia. Passive transfer demonstrated that immune IgM from AID-deficient mice contributes to Friend virus control in the presence of virus-specific CD4+ T cells.

Interleukin-1-induced acute bone resorption facilitates the secretion of fibroblast growth factor 23 into the circulation.

Fibroblast growth factor 23 (FGF23), a central regulator of phosphate and vitamin D metabolism, is mainly produced by osteocytes in bone and exerts its effects on distant organs. Despite its endocrine function, the mechanism controlling serum FGF23 levels is not fully understood. Here we tested the hypothesis that osteoclastic bone resorption may play a role in regulating circulating levels of FGF23, using a mouse model where injections of interleukin (IL)-1ß into the subcutaneous tissue over the calvaria induced rapid bone resorption. A significant amount of FGF23 was detected in the extracts from mouse bones, which supports the idea that FGF23 stays in bone for a while after its production. IL-1ß-induced bone resorption was associated with elevated serum FGF23 levels, an effect abolished by pre-treatment with pamidronate. Fgf23 expression was not increased in either the calvariae or tibiae of IL-1ß-injected mice, which suggests that IL-1ß facilitated the entry of FGF23 protein into circulation by accelerating bone resorption rather than increasing its gene expression. The direct effect of IL-1ß on bone was confirmed when it increased FGF23 levels in the conditioned media of mouse calvariae in organ culture. Repeated treatment of the cultured calvariae with IL-1ß led to a refractory phase, where FGF23 was not mobilized by IL-1ß anymore. Consistent with the in vivo results, treatment with IL-1ß failed to increase Fgf23 mRNA in isolated primary osteocytes and osteoblasts. These results suggest that FGF23 produced by osteocytes remains in bone, and that rapid bone resorption facilitates its entry into the bloodstream.

Infection of adult thymus with murine retrovirus induces virus-specific central tolerance that prevents functional memory CD8+ T cell differentiation.

In chronic viral infections, persistent antigen presentation causes progressive exhaustion of virus-specific CD8+ T cells. It has become clear, however, that virus-specific naïve CD8+ T cells newly generated from the thymus can be primed with persisting antigens. In the setting of low antigen density and resolved inflammation, newly primed CD8+ T cells are preferentially recruited into the functional memory pool. Thus, continual recruitment of naïve CD8+ T cells from the thymus is important for preserving the population of functional memory CD8+ T cells in chronically infected animals. Friend virus (FV) is the pathogenic murine retrovirus that establishes chronic infection in adult mice, which is bolstered by the profound exhaustion of virus-specific CD8+ T cells induced during the early phase of infection. Here we show an additional evasion strategy in which FV disseminates efficiently into the thymus, ultimately leading to clonal deletion of thymocytes that are reactive to FV antigens. Owing to the resultant lack of virus-specific recent thymic emigrants, along with the above exhaustion of antigen-experienced peripheral CD8+ T cells, mice chronically infected with FV fail to establish a functional virus-specific CD8+ T cell pool, and are highly susceptible to challenge with tumor cells expressing FV-encoded antigen. However, FV-specific naïve CD8+ T cells generated in uninfected mice can be primed and differentiate into functional memory CD8+ T cells upon their transfer into chronically infected animals. These findings indicate that virus-induced central tolerance that develops during the chronic phase of infection accelerates the accumulation of dysfunctional memory CD8+ T cells.

Sympathetic activation induces skeletal Fgf23 expression in a circadian rhythm-dependent manner.

The circadian clock network is well known to link food intake and metabolic outputs. Phosphorus is a pivotal nutritional factor involved in energy and skeletal metabolisms and possesses a circadian profile in the circulation; however, the precise mechanisms whereby phosphate metabolism is regulated by the circadian clock network remain largely unknown. Because sympathetic tone, which displays a circadian profile, is activated by food intake, we tested the hypothesis that phosphate metabolism was regulated by the circadian clock network through the modification of food intake-associated sympathetic activation. Skeletal Fgf23 expression showed higher expression during the dark phase (DP) associated with elevated circulating FGF23 levels and enhanced phosphate excretion in the urine. The peaks in skeletal Fgf23 expression and urine epinephrine levels, a marker for sympathetic tone, shifted from DP to the light phase (LP) when mice were fed during LP. Interestingly, ß-adrenergic agonist, isoproterenol (ISO), induced skeletal Fgf23 expression when administered at ZT12, but this was not observed in Bmal1-deficient mice. In vitro reporter assays revealed that ISO trans-activated Fgf23 promoter through a cAMP responsive element in osteoblastic UMR-106 cells. The mechanism of circadian regulation of Fgf23 induction by ISO in vivo was partly explained by the suppressive effect of Cryptochrome1 (Cry1) on ISO signaling. These results indicate that the regulation of skeletal Fgf23 expression by sympathetic activity is dependent on the circadian clock system and may shed light on new regulatory networks of FGF23 that could be important for understanding the physiology of phosphate metabolism.

Differential requirements of cellular and humoral immune responses for Fv2-associated resistance to erythroleukemia and for regulation of retrovirus-induced myeloid leukemia development.

To assess the possible contribution of host immune responses to the exertion of Fv2-associated resistance to Friend virus (FV)-induced disease development, we inoculated C57BL/6 (B6) mice that lacked various subsets of lymphocytes with FV containing no lactate dehydrogenase-elevating virus. Fv2(r) B6 mice lacking CD4(+) T cells developed early polycythemia and fatal erythroleukemia, while B6 mice lacking CD8(+) T cells remained resistant. Erythroid progenitor cells infected with spleen focus-forming virus (SFFV) were eliminated, and no polycythemia was observed in B cell-deficient B6 mice, but they later developed myeloid leukemia associated with oligoclonal integration of ecotropic Friend murine leukemia virus. Additional depletion of natural killer and/or CD8(+) T cells from B cell-deficient B6 mice resulted in the expansion of SFFV proviruses and the development of polycythemia, indicating that SFFV-infected erythroid cells are not only restricted in their growth but are actively eliminated in Fv2(r) mice through cellular immune responses.

FGF23 suppresses chondrocyte proliferation in the presence of soluble α-Klotho both in vitro and in vivo.

Fibroblast growth factor-23 (FGF23) is well established to play crucial roles in the regulation of phosphate homeostasis. X-linked hypophosphatemic rickets (XLH) is characterized by impaired mineralization and growth retardation associated with elevated circulating FGF23 levels. Administration of phosphate and calcitriol is effective in improving growth retardation, but is not sufficient to fully reverse impaired growth, suggesting the existence of a disease-specific mechanism in the development of growth retardation in addition to dysregulated phosphate metabolism. However, the precise mechanisms of growth retardation in XLH remain elusive. Here, we postulated that FGF23 suppressed chondrocyte proliferation in the presence of soluble α-Klotho (sKL). In vitro and ex vivo studies revealed that FGF23 formed a protein complex with sKL through KL1 internal repeat and suppressed the linear growth of metatarsals in the presence of sKL, which was antagonized by co-incubation with neutralizing antibodies against FGF23 or by knocking-down FGFR3 expression. Additionally, FGF23 binding to FGFR3 was enhanced in the presence of sKL. Histologically, the length of the proliferating zone was diminished and was associated with decreased chondrocyte proliferation. FGF23/sKL suppressed Indian hedgehog (Ihh) expression and administration of Ihh protein partially rescued the suppressive effect of FGF23/sKL on metatarsal growth. Intraperitoneal administration of sKL in Hyp mice, a murine model for XLH, caused a decrease in the length of the proliferating zone associated with decreased chondrocyte proliferation without altering circulating phosphate levels. These findings suggest that suppression of chondrocyte proliferation by FGF23 could have a causative role in the development of growth retardation in XLH.

Natural killer cells recognize friend retrovirus-infected erythroid progenitor cells through NKG2D-RAE-1 interactions In Vivo.

Natural killer (NK) cells function as early effector cells in the innate immune defense against viral infections and also participate in the regulation of normal and malignant hematopoiesis. NK cell activities have been associated with early clearance of viremia in experimental simian immunodeficiency virus and clinical human immunodeficiency virus type 1 (HIV-1) infections. We have previously shown that NK cells function as major cytotoxic effector cells in vaccine-induced immune protection against Friend virus (FV)-induced leukemia, and NK cell depletion totally abrogates the above protective immunity. However, how NK cells recognize retrovirus-infected cells remains largely unclear. The present study demonstrates a correlation between the expression of the products of retinoic acid early transcript-1 (RAE-1) genes in target cells and their susceptibility to killing by NK cells isolated from FV-infected animals. This killing was abrogated by antibodies blocking the NKG2D receptor in vitro. Further, the expression of RAE-1 proteins on erythroblast surfaces increased early after FV inoculation, and administration of an RAE-1-blocking antibody resulted in increased spleen infectious centers and exaggerated pathology, indicating that FV-infected erythroid cells are recognized by NK cells mainly through the NKG2D-RAE-1 interactions in vivo. Enhanced retroviral replication due to host gene-targeting resulted in markedly increased RAE-1 expression in the absence of massive erythroid cell proliferation, indicating a direct role of retroviral replication in RAE-1 upregulation.

Attenuated food anticipatory activity and abnormal circadian locomotor rhythms in Rgs16 knockdown mice.

Regulators of G protein signaling (RGS) are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs) of G protein α-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN), the master circadian light-entrainable oscillator (LEO) of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA) targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO)-driven elevated food-anticipatory activity (FAA) observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s).

Persistence of viremia and production of neutralizing antibodies differentially regulated by polymorphic APOBEC3 and BAFF-R loci in friend virus-infected mice.

Several host genes control retroviral replication and pathogenesis through the regulation of immune responses to viral antigens. The Rfv3 gene influences the persistence of viremia and production of virus-neutralizing antibodies in mice infected with Friend mouse retrovirus complex (FV). This locus has been mapped within a narrow segment of mouse chromosome 15 harboring the APOBEC3 and BAFF-R loci, both of which show functional polymorphisms among different strains of mice. The exon 5-lacking product of the APOBEC3 allele expressed in FV-resistant C57BL/6 (B6) mice directly restricts viral replication, and mice lacking the B6-derived APOBEC3 exhibit exaggerated pathology and reduced production of neutralizing antibodies. However, the mechanisms by which the polymorphisms at the APOBEC3 locus affect the production of neutralizing antibodies remain unclear. Here we show that the APOBEC3 genotypes do not directly affect the B-cell repertoire, and mice lacking B6-derived APOBEC3 still produce FV-neutralizing antibodies in the presence of primed T helper cells. Instead, higher viral loads at a very early stage of FV infection caused by either a lack of the B6-derived APOBEC3 or a lack of the wild-type BAFF-R resulted in slower production of neutralizing antibodies. Indeed, B cells were hyperactivated soon after infection in the APOBEC3- or BAFF-R-deficient mice. In contrast to mice deficient in the B6-derived APOBEC3, which cleared viremia by 4 weeks after FV infection, mice lacking the functional BAFF-R allele exhibited sustained viremia, indicating that the polymorphisms at the BAFF-R locus may better explain the Rfv3-defining phenotype of persistent viremia.

Increased liver temperature efficiently augments human cellular immune response: T-cell activation and possible monocyte translocation.

Hyperthermia (HT), in combination with other conventional therapeutic modalities, has become a promising approach in cancer therapy. In addition to heat-induced apoptosis, an augmented immunological effect is considered to be a benefit of hyperthermic treatment over chemo- or radiotherapy. Here, we investigated the effect of regional HT targeting the liver on immune cells, especially T cells and antigen-presenting cells, which are important in recognizing and eliminating tumor cells and pathogens such as viruses. In healthy volunteers exposed to such regional HT, both CD4(+) and CD8(+) T cells that express an activation marker CD69 increased transiently at 1 h post-treatment, with a subsequent decrease to base levels at 6 h after the treatment. At 24 h post-treatment, the percentage of CD69-positive cells significantly increased again but only among CD8(+) T cells. IFN-gamma production from PHA-stimulated peripheral blood mononuclear cells was gradually and significantly increased in the 2 days following the heating procedure, peaking at 36 h post-treatment. Furthermore, we found marked increases in plasma levels of IL-1beta and IL-6 starting at 24 h post-treatment. With regard to the number of each leukocyte subpopulation, a transient and dramatic decrease in the number of a subset of monocytes, CD14(+) CD16(-) cells, was observed at 1 h after the hyperthermic treatment, suggesting that the regional HT aimed at the liver may have influenced the extravasation of blood monocytes. No significant changes in T-cell activities or monocyte counts were observed in the volunteers exposed to heating of the lungs or the legs. These results suggest that heating of the liver may efficiently induce cellular immune responses to liver cancers.

Diethyldithiocarbamate can induce two different type of death: apoptosis and necrosis mediating the differential MAP kinase activation and redox regulation in HL60 cells.

Although apoptosis and necrosis have been considered different pathways to cell death, only one compound induces both types of cell death. Diethyldithiocarbamate (DDC) has been shown to have antioxidant or prooxidant effects in several different systems. We observed in our present study that DDC induced not only apoptosis but also necrosis depending on its dosage in HL60 premyelocytic leukemia cells. Moreover, in hypoxia cell culture conditions, DDC-induced necrotic cells decreased but DDC-induced apoptosis continued. We investigated the DDC-induced different cell death mechanisms as they are correlated with reactive oxygen species (ROS). High-dose DDC-induced necrotic cell death is thought to depend on the increase of intracellular ROS, while low-dose DDC-induced apoptosis is thought to depend on changes of the intracellular redox state by the transporting of external metal ions. There was no sequential or quantitative change of Bcl-2 family proteins in DDC-induced apoptotic or necrotic pathways. However, the mitochondrial transmembrane potential was remarkably decreased in the DDC-induced necrosis. Finally, duration of c-Jun N-terminal kinase (JNK) activation resulted in different types of cell death.

Type IV collagen, type IV collagenase activity and ability of cell proliferation in human thyroid tumours.

BACKGROUND: The processes of malignant tumour invasion and metastasis are known to include the destruction of cell stroma and vascular basement membrane. It has been suggested that type IV collagenase degrades type IV collagen, a main component of the basement membrane. METHODS: In our study, type IV collagenase activity in human thyroid tumours was measured by the Liotta method. The degree of destruction of diseased regions of thyroid tumours was immunohistochemically determined by anti-type IV collagen antibody staining. Cell proliferation in the tumours was estimated using anti-proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGFR). RESULTS: T4 thyroid carcinomas with higher type IV collagenase activity and very weak type IV discontinuous immunostaining for type IV collagen of follicular basement membranes, exhibited many PCNA or EGFR positive cells. In benign tumours, normofollicular- or macrofollicular-type tumours with low type IV collagenase activity showed few PCNA and EGFR positive cells and intact type IV collagen of basement membranes, as seen in normal thyroids. Conversely, an atypical adenoma with higher type IV collagenase activity showed many PCNA and EGFR positive cells and weak type IV discontinuous immunostaining for type IV collagen, as in thyroid carcinomas. CONCLUSION: These findings suggest that staining for type IV collagen and type IV collagenase activity reflect the ability of cell proliferation, and help predict the aggressiveness of invasion and metastasis in human thyroid tumours.

Change of Cu,Zn-superoxide dismutase activity of guinea pig lung in experimental asthma.

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.