Characterization of the symbiont Rickettsia in the mirid bug Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae).

Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae) is an omnivorous insect used for biological control. Augmentative release and conservation of N. tenuis have been used for pest control in tomato crops. Intracellular bacterial symbionts of arthropods are common in nature and have diverse effects on their hosts; in some cases they can dramatically affect biological control. Fingerprinting methods showed that the symbiotic complex associated with N. tenuis includes Wolbachia and Rickettsia. Rickettsia of N. tenuis was further characterized by sequencing the 16S rRNA and gltA bacterial genes, measuring its amount in different developmental stages of the insect by real-time polymerase chain reaction, and localizing the bacteria in the insect's body by fluorescence in situ hybridization. The Rickettsia in N. tenuis exhibited 99 and 96% similarity of both sequenced genes to Rickettsia bellii and Rickettsia reported from Bemisia tabaci, respectively. The highest amount of Rickettsia was measured in the 5th instar and adult, and the symbionts could be detected in the host gut and ovaries. Although the role played by Rickettsia in the biology of N. tenuis is currently unknown, their high amount in the adults and localization in the gut suggest that they may have a nutritional role in this insect.

Healthcare contacts with self-harm during COVID-19: An e-cohort whole-population-based study using individual-level linked routine electronic health records in Wales, UK, 2016-March 2021.

INTRODUCTION: Reduced rates of help seeking by those who self-harmed during the COVID-19 pandemic have been reported. OBJECTIVES: To understand changes in healthcare service contacts for self-harm during the COVID-19 pandemic across primary, emergency and secondary care. METHODS: This retrospective cohort study used routine electronic healthcare data for Wales, United Kingdom, from 2016 to March 14, 2021. Population-based data from primary care, emergency departments and hospital admissions were linked at individual-level. All Welsh residents aged ≥10 years over the study period were included in the study. Primary, emergency and secondary care contacts with self-harm at any time between 2016 and March 14, 2021 were identified. Outcomes were counts, incidence, prevalence and proportion of self-harm contacts relative to all contacts in each and all settings, as well as the proportion of people contacting one or more settings with self-harm. Weekly trends were modelled using generalised estimated equations, with differences between 2020 (to March 2021) and comparison years 2016-2018 (to March 2017-2019) quantified using difference in differences, from which mean rate of odds ratios (µROR) across years was reported. RESULTS: The study included 3,552,210 individuals over the study period. Self-harm contacts reduced across services in March and December 2020 compared to previous years. Primary care contacts with self-harm reduced disproportionately compared to non-self-harm contacts (µROR = 0.7, p<0.05), while their proportion increased in emergency departments during April 2020 (µROR = 1.3, p<0.05 in 2/3 comparison years) and hospital admissions during April-May 2020 (µROR = 1.2, p<0.05 in 2/3 comparison years). Despite this, those who self-harmed in April 2020 were more likely to be seen in primary care than other settings compared to previous years (µROR = 1.2, p<0.05). A lower proportion of those with self-harm contacts in emergency departments were subsequently admitted to hospital in December 2020 compared to previous years (µROR = 0.5, p<0.05). CONCLUSIONS: These findings suggest that those who self-harmed during the COVID-19 pandemic may have been less likely to seek help, and those who did so faced more stringent criteria for admission. Communications encouraging those who self-harm to seek help during pandemics may be beneficial. However, this needs to be supported by maintained provision of mental health services.

Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis.

Heparan sulfate proteoglycans interact with many extracellular matrix constituents, growth factors and enzymes. Degradation of heparan sulfate by endoglycosidic heparanase cleavage affects a variety of biological processes. We have purified a 50-kDa heparanase from human hepatoma and placenta, and now report cloning of the cDNA and gene encoding this enzyme. Expression of the cloned cDNA in insect and mammalian cells yielded 65-kDa and 50-kDa recombinant heparanase proteins. The 50-kDa enzyme represents an N-terminally processed enzyme, at least 100-fold more active than the 65-kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and specimens of human breast, colon and liver carcinomas. Low metastatic murine T-lymphoma and melanoma cells transfected with the heparanase cDNA acquired a highly metastatic phenotype in vivo, reflected by a massive liver and lung colonization. This represents the first cloned mammalian heparanase, to our knowledge, and provides direct evidence for its role in tumor metastasis. Cloning of the heparanase gene enables the development of specific molecular probes for early detection and treatment of cancer metastasis and autoimmune disorders.

Counteracting age-related VEGF signaling insufficiency promotes healthy aging and extends life span.

Aging is an established risk factor for vascular diseases, but vascular aging itself may contribute to the progressive deterioration of organ function. Here, we show in aged mice that vascular endothelial growth factor (VEGF) signaling insufficiency, which is caused by increased production of decoy receptors, may drive physiological aging across multiple organ systems. Increasing VEGF signaling prevented age-associated capillary loss, improved organ perfusion and function, and extended life span. Healthier aging was evidenced by favorable metabolism and body composition and amelioration of aging-associated pathologies including hepatic steatosis, sarcopenia, osteoporosis, "inflammaging" (age-related multiorgan chronic inflammation), and increased tumor burden. These results indicate that VEGF signaling insufficiency affects organ aging in mice and suggest that modulating this pathway may result in increased mammalian life span and improved overall health.

Hox genes in normal and neoplastic mouse mammary gland.

Homeobox-containing genes regulate embryonic developmental programs and are expressed in certain adult tissues and cancers. There has been no report of expression in the breast. We amplified homeobox complementary DNA from mouse mammary gland and found expression of members from each of the four major Hox gene clusters. The regulation of expression of two Hox genes was examined in greater depth. Hoxc-6 transcripts were present in the glands of pubescent and mature mice and decreased during pregnancy. Levels were increased substantially following ovariectomy, indicating possible negative regulation by steroid hormones. Hox expression was studied in mammary adenocarcinomas and in transplant lines of the benign, precancerous tissues from which the cancers arose. Hoxc-6 was expressed at low levels in the precancerous tissue but was not expressed in cancers. In contrast, Hoxa-1 was expressed only in cancers, not in normal gland or in precancerous mammary tissues, suggesting that Hox genes may play a role in a late stage in the stepwise development of mammary malignancies.

Molecular properties and involvement of heparanase in cancer progression and normal development.

Heparan sulfate proteoglycans (HSPGs) play a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Hence, cleavage of heparan sulfate (HS) affects the integrity and functional state of tissues and thereby fundamental normal and pathological phenomena involving cell migration and response to changes in the extracellular microenvironment. Here, we describe the molecular properties, expression and function of a human heparanase, degrading HS at specific intrachain sites. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and human tumor tissues. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also releases ECM-resident angiogenic factors in vitro and its overexpression induces an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, both critical steps in cancer progression. The enzyme is also involved in cell migration associated with inflammation and autoimmunity. The unexpected identification of a single predominant functional heparanase suggests that the enzyme is a promising target for drug development. In fact, treatment with heparanase inhibitors markedly reduces tumor growth, metastasis and autoimmune disorders in animal models. Studies are underway to elucidate the involvement of heparanase in normal processes such as implantation, embryonic development, morphogenesis, tissue repair, inflammation and HSPG turnover. Heparanase is the first functional mammalian HS-degrading enzyme that has been cloned, expressed and characterized. This may lead to identification and cloning of other glycosaminoglycan degrading enzymes, toward a better understanding of their involvement and significance in normal and pathological processes.

Expression of heparanase, Mdm2, and erbB2 in ovarian cancer.

Ovarian cancer is the most lethal of gynecological malignancies. Yet early diagnosis and prognosis are far from being satisfactory. Degradation of heparan sulfate proteoglycans by heparanase appears to play an important role in the invasiveness of tumor cells through the basement membrane and into the extracellular matrix. Recent cloning of the heparanase gene and generation of monoclonal antibodies against the enzyme permit to examine tumor cell expression of the enzyme. The aim of the present study was to assess heparanase activity and localization in various subtypes of epithelial ovarian cancer in correlation with oncogene expression. Histologically confirmed malignant ovarian tissue from ten women and tissue from 2 benign ovarian tumors and 4 normal ovaries were assessed for heparanase presence, activity and localization, incidence of apoptosis and expression of the oncogenes erbB2 and Mdm2. Heparanase immunohistostaining and activity were present in mucinous carcinomas and were more intense than in endometrioid and in serous carcinomas. The lowest activity was observed in benign ovarian tumors and normal ovaries. In ovarian carcinomas the enzyme was intensely concentrated in the cytoplasm of the cancerous cells. In contrast, in normal ovaries and benign tumors the enzyme was predominantly localized in endothelial cells lining blood capillaries. The rate of apoptosis was considerably higher in mucinous and endometrioid carcinomas, and was lower in serous and primary peritoneal carcinomas. Extremely high concentration of heparanase was often demonstrated in apoptotic cells. Endometrioid and serous carcinomas showed high expression of Mdm2 and erbB2 while mucinous carcinomas showed low expression. In benign ovarian tumors and normal ovaries the expression of both oncoproteins was extremely low. In conclusion ovarian carcinomas demonstrate higher levels of heparanase than benign tumors and normal ovaries suggesting that the enzyme may play an important role in metastatic spread of the cancerous cells. Apoptosis may be a significant part of the mechanism of the enzyme release into the extracellular space. Although heparanase activity seems to play an essential role in tumor progression, expression of oncogenes, such as erbB2 and Mdm2 seems to play the dominant role in the development of ovarian cancer.

Mammalian heparanase as mediator of tumor metastasis and angiogenesis.

Expression of heparan sulfate-degrading endoglycosidases, commonly referred to as heparanases, correlates with the metastatic potential of tumor cell lines, and treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. We purified a 50 kDa heparanase from human hepatoma and placenta and cloned a cDNA and gene encoding a protein of 543 amino acids. Only one heparanase sequence was identified, suggesting that this enzyme is the dominant endoglucuronidase in mammalian tissues. Expression of the cloned cDNA in insect and mammalian cells yielded 65 kDa and 50 kDa recombinant proteins. The 50 kDa enzyme represents an N-terminal processed enzyme that is at least 200-fold more active than the full-length 65 kDa form. Processing was demonstrated following incubation of the full-length recombinant enzyme with intact tumor cells. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and in specimens of human melanomas and carcinomas. In the colon, both the heparanase mRNA and protein are expressed already at the stage of tubulovillous adenoma, but not in the adjacent 'normal-looking' colon epithelium. Non-metastatic murine T lymphoma and melanoma cells transfected with the heparanase gene acquired a highly metastatic phenotype in vivo. Apart from its involvement in the egress of cells from the vasculature, heparanase is tightly involved in angiogenesis, both directly--by promoting invasion of endothelial cells (vascular sprouting), and indirectly--by releasing heparan sulfate-bound basic fibroblast growth factor, and generating HS degradation fragments that promote bFGF activity. The angiogenic potential of heparanase was demonstrated in vivo (Matrigel plug assay) by showing a three to fourfold increase in neovascularization induced by Eb T lymphoma cells following their transfection with the heparanase gene. The ability of heparanase to promote both tumor angiogenesis and metastasis makes it a promising target for cancer therapy.

Regulated expression of homeobox genes Msx-1 and Msx-2 in mouse mammary gland development suggests a role in hormone action and epithelial-stromal interactions.

The murine homeobox genes Msx-1 and Msx-2 are related to the Drosophila msh gene and are expressed in a variety of tissues during mouse embryogenesis. We now report the developmentally regulated expression of Msx-1 and Msx-2 in the mouse mammary gland and show that their expression patterns point toward significant functional roles. Msx-1 and Msx-2 transcripts were present in glands of virgin mice and in glands of mice in early pregnancy, but transcripts decreased dramatically during late pregnancy. Low levels of Msx-1 transcripts were detected in glands from lactating animals and during the first days of involution, whereas Msx-2 expression was not detected during lactation or early involution. Expression of both genes increased gradually as involution progressed. Msx-2 but not Msx-1 expression was decreased following ovariectomy or following exposure to anti-estrogen implanted directly into the gland. Hormonal regulation of Msx-2 expression was confirmed when transcripts returned to normal levels after estrogen was administered to ovariectomized animals. In situ molecular hybridization for Msx-1 showed transcripts localized to the mammary epithelium, whereas Msx-2 expression was confined to the periductal stroma. Mammary stroma from which mammary epithelium had been removed did not transcribe detectable amounts of Msx-2, showing that expression is regulated by contiguous mammary epithelium, and indicating a role for these homeobox genes in mesenchymal-epithelial interactions during mammary development.

Expression and functional role of E- and P-cadherins in mouse mammary ductal morphogenesis and growth.

Mammary ducts, and the highly mitotic terminal end buds from which they are derived, consist of two layers of ectodermally derived epithelium, forming a tube-within-a-tube structure. We investigated the role of Ca(2+)-dependent cell-cell adhesion molecules in maintaining the integrity of these layers. Immunostaining showed abundant E-cadherin on the lateral membranes of end bud body cells and ductal lumenal cells, but no P-cadherin. The basally located cap cells and their differentiated descendants, the ductal myoepithelial cells, displayed only P-cadherin. We investigated the functional significance of this pattern of cadherin expression in situ by surgically implanting small, slow-release plastic implants releasing function-blocking antibodies. End buds exposed to a monoclonal antibody to E-cadherin showed disruption of the body epithelium, with epithelial cells floating freely in the lumen. Epithelial DNA synthesis, which is normally very high in these growth buds, abruptly declined. That this reduction in growth was not due to cell damage was shown by spontaneous reaggregation of the cells into a normal epithelium, with resumption of DNA synthesis, when the blocking antibody was depleted. A monoclonal antibody to P-cadherin had no effect on the lumenal layer but partially disrupted the basally located cap cell layer. These data indicate that spatially selective expression of E- and P-cadherins is required for mammary tissue integrity, which is in turn a prerequisite for normal rates of DNA synthesis.

Megakaryocyte maturation is associated with expression of the CXC chemokine connective tissue-activating peptide CTAP III.

Connective tissue-activating peptide III (CTAP III), a CXC chemokine derived from the chemokine precursor platelet basic protein (PBP) by proteolytic cleavage, has been identified in platelets, activated macrophages, neutrophils and T lymphocytes. CTAP III can support stem cell-derived haematopoiesis yet inhibits the proliferation of committed megakaryocyte (MK) progenitors. This investigation was aimed at characterizing CTAP III expression in human MKs and determining it's role in MK differentiation. We report high expression of CTAP III in mature human bone marrow (BM) MKs and megakaryoblast cell lines following differentiation induction with phorbol ester 12-myristate 13-acetate (PMA). Immunostaining with anti-CTAP III antibodies demonstrated its prominent presence in platelet producing zones in the cytoplasm and intense staining around the periphery of the large BM MKs. In cultures of logarithmically growing megakaryoblast cell lines DAMI, CHRF-288 or MEG01, which contain primarily 2N cells, only 15% of the cells expressed CTAP III. The addition of PMA stimulated high levels of CTAP III after 24 h in more than 75% of the cells, being expressed in both the 2N and large polyploid MKs. Reverse transcription polymerase chain reaction (RT-PCR) revealed upregulation of CTAP III mRNA after only 1 h of exposure to PMA that was sustained for 24 h. In the bone marrow of idiopathic thrombocytopenic pupura (ITP) patients undergoing accelerated MK maturation and thrombopoiesis, 99% of large MKs and 95% of small MKs expressed high levels of CTAP III. While the biological function of this chemokine in MKs is not known, these studies demonstrate that molecular upregulation of CTAP III in MKs is associated with maturation and, as with other chemokines, may be involved in proliferation arrest and cellular interactions with extracellular matrix and platelet production.

Expression of heparanase in normal, dysplastic, and neoplastic human colonic mucosa and stroma. Evidence for its role in colonic tumorigenesis.

The human heparanase gene, an endo-beta-glucuronidase that cleaves heparan sulfate at specific intrachain sites, has recently been cloned and shown to function in tumor progression and metastatic spread. Antisense digoxigenin-labeled heparanase RNA probe and monoclonal anti-human heparanase antibodies were used to examine the expression of the heparanase gene and protein in normal, dysplastic, and neoplastic human colonic mucosa. To our knowledge, this is the first systematic study of heparanase expression in human colon cancer. Both the heparanase gene and protein were expressed at early stages of neoplasia, already at the stage of adenoma, but were practically not detected in the adjacent normal-looking colon epithelium. Gradually increasing expression of heparanase was evident as the cells progressed from severe dysplasia through well-differentiated to poorly differentiated colon carcinoma. Deeply invading colon carcinoma cells showed the highest levels of the heparanase mRNA and protein associated with expression of both the gene and enzyme by adjacent desmoplastic stromal fibroblasts. A high expression was also found in colon carcinoma metastases to lung, liver, and lymph nodes, as well as in the accompanying stromal fibroblasts. Moreover, extracts derived from tumor tissue expressed much higher levels of the heparanase protein and activity as compared to the normal colon tissue. In all specimens, the heparanase gene and protein exhibited the same pattern of expression. These results suggest a role of heparanase in colon cancer progression and may have both prognostic and therapeutic applications.

Expression of Hoxa-1 and Hoxb-7 is regulated by extracellular matrix-dependent signals in mammary epithelial cells.

Homeobox-containing genes encode transcriptional regulators involved in cell fate and pattern formation during embryogenesis. Recently, it has become clear that their expression in continuously developing adult tissues, as well as in tumorigenesis, may be of equal importance. In the mouse mammary gland, expression patterns of several homeobox genes suggest a role in epithelial-stromal interactions. Because the stroma and the extracellular matrix (ECM) are known to influence both functional and morphological development of the mammary gland, we asked whether these genes would be expressed postnatally in the gland and also in cell lines in culture and whether they could be modulated by ECM. Using a polymerase chain reaction-base strategy five members of the Hox gene clusters a and b were shown to be expressed in cultured mouse mammary cells. Hoxa-1 and Hoxb-7 were chosen for further analysis. Hoxb-7 was chosen because it had not been described previously in the mammary gland and was modulated at different stages of gland development. Hoxa-1 was chosen because it was reported previously to be expressed only in mammary tumors, and not in normal glands. We showed that culturing the mammary epithelial cell lines SCp2 and CID-9 on a basement membrane (BM) that was previously shown to induce a lactational phenotype was necessary to turn off Hoxb-7, but a change in cell shape, brought about by culturing the cells on an inert substratum such as polyHEMA, was sufficient to downregulate Hoxa-1. This is the first report of modulation of homeobox genes by ECM. The results provide a rationale for the differential pattern of expression in vivo of Hoxa-1 and Hoxb-7 during different stages of development. The culture model should permit further in-depth analysis of the molecular mechanisms involved in how ECM signaling and homeobox genes may interact to bring about tissue organization.

Connective tissue activating peptide III expression disappears progressively with increased dysplasia in human cervical epithelium.

OBJECTIVE: The aim of this study was to explore the possible involvement of CTAP-III in the development of cervical cancer as it progresses through several cervical intraepithelial neoplasia (CIN) stages. MATERIAL AND METHODS: Twenty-four cervical specimens were obtained by direct punch biopsy, conization, or hysterectomy. Diagnosis of CIN I to CIN III was based on standard morphological criteria in 12 specimens. Tissue specimens were also obtained from 4 normal uteri and 8 cases of invasive squamous cell cervical carcinoma. RT-PCR, using CTAP-III-specific primers, was used to identify CTAP-III mRNA and polyclonal antibodies, directed against the N-terminus of CTAP-III, for immunostaining of the CTAP-III protein. RESULTS: RT-PCR yielded amplified fragments in RNA derived from normal cervical tissue, while no PCR product was detected in the invasive cervical carcinoma tissue. The PCR product corresponded to a CTAP-III plasmid PCR product. Both tissues expressed the same amounts of GAPDH mRNA as the control for the integrity and equal amounts of the isolated RNA. In each of the 16 specimens of normal cervices and of CIN tissues, epithelial cells were stained with the anti-CTAP-III antibodies. In normal epithelium, CTAP-III staining was homogeneously distributed in all epithelial layers, except in the highly active and proliferating basal cells. CTAP-III was localized at the epithelial cell membrane or between adjacent epithelial cells in a granular, chain-like pattern of staining. In the CIN specimens, CTAP-III staining was no longer seen in the deep epithelial layers, consistent with the dysplastic appearance of the cells, and remained in the seemingly normal superficial epithelial layers. Cells of invasive cervical carcinoma did not stain for CTAP-III and were detected in endothelial cells of capillary blood vessels. CONCLUSION: The specific localization of CTAP-III between adjacent epithelial cells suggests a possible role of this chemokine in maintaining the normal architecture of epithelial tissues. Its progressive disappearance in increasingly severe CIN may be applied to distinguish between specific stages in the progression of cervical carcinoma.

Molecular properties and involvement of heparanase in cancer progression and mammary gland morphogenesis.

Tumor spread involves degradation of various components of the extracellular matrix and blood vessel wall. Among these is heparan sulfate proteoglycan, which plays a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Expression of an endoglycosidase (heparanase) which degrades heparan sulfate correlates with the metastatic potential of tumor cells, and treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. Heparin-binding angiogenic proteins are stored as a complex with heparan sulfate in the microenvironment of tumors. These proteins are released and can induce new capillary growth when heparan sulfate is degraded by heparanase. Here, we describe the molecular properties, expression and involvement in tumor progression of a human heparanase. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic human cell lines and in tumor biopsy specimens, including breast carcinoma. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also released ECM-resident bFGF in vitro, and its overexpression elicited an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, two critical steps in tumor progression. Mammary glands of transgenic mice overexpressing the heparanase enzyme exhibit precocious branching of ducts and alveolar development, suggesting that the enzyme promotes normal morphogenesis and possibly pre-malignant changes in the mammary gland.