Genomic Function and Variation of Human Polyomavirus BK (BKV).
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Journal Article↗
Biomedical subjects
L Jin
Publications and source records attributed to L Jin.
Development of a dual target-PCR for detection and characterization of measles virus in clinical specimens.
A polymerase chain reaction (PCR) method was developed for detecting measles virus (MV) RNA in a variety of clinical samples using primer pairs in the nucleocapsid (N) and matrix (M) genes in one reaction (dual target-PCR). The dual target-PCR detected MV RNA in tissue culture fluid containing 1 TCID50 of the MV Loss strain, and was as sensitive as a single target-PCR. Specificity was confirmed by the failure of the dual target-PCR to amplify products from the infected tissue culture containing related paramyxoviruses. Thirty-two of 35 (91.4%) samples collected from 23 patients with confirmed measles infection by the detection of measles specific IgM were found to be positive for MV RNA by PCR. Direct sequencing of the PCR amplicons revealed three different genotypes among the MV strains that were detected in 12 patients. The dual target-PCR method is suitable for the diagnosis of measles infection and, based on the sequencing of the PCR product DNA, for investigating the molecular epidemiology of MV strains.
Adult↗
Localization of zinc and metallothionein in the rat bladder epithelium during carcinogenesis induced by N-butyl-N-(4-hydroxybutyl) nitrosamine.
This study investigated the presence of zinc and expression of metallothionein (MT) in different pathological changes of the rat bladder induced by administration of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Using the Timm staining method, the presence of zinc was observed in normal and benign hyperplastic epithelial cells of the rat bladder, particularly in the malignant bladder tumor, induced by the administration of BBN. Immunohistochemically, MT expression was detected only in noninvasive (30%) and invasive transitional cell carcinoma (80%) of the rat bladder where the tumor cells were rich in zinc. Our data suggest that: (1) growth and development of the rat bladder tumor, especially malignant tumors, may have a high requirement for zinc and (2) MT synthesis may be induced by a high zinc concentration in rat bladder tumor cells.
Animals↗
A chronological study of the expression of glial fibrillary acidic protein and calbindin-D28 k by reactive astrocytes in the electrically lesioned rat brain.
Immunoreactivity of neuronal and glial marker proteins of reactive astrocytes around the electrically damaged pyramidal layer and stratum radiatum of the hippocampal CA1 region and corpus callosum was chronologically studied in electrically lesioned rat brains. A monoclonal antibody against calbindin-D28 k (CD28-Ab) and a polyclonal antibody against glial fibrillary acidic protein (GFAP-Ab) were used for immunostaining. Immunoreactivity of CD28 and GFAP in the reactive astrocytes was detected in brains 1-6 weeks post-lesion but not in non-lesioned brains. The number of immunohistochemically stained reactive astrocytes around the electrically damaged areas were counted and then compared with the number of those in the same areas of non-lesioned brains. The number of CD28- and GFAP-immunoreactive astrocytes began to increase around the lesion from 1-3 weeks following lesion in the pyramidal layer of the hippocampal CA1 region and from 1-4 weeks following lesion in the stratum radiatum of the hippocampal CA1 region and corpus callosum. These immunoreactive astrocytes could be observed for 6 weeks (the maximum survival time studies) in all areas of the lesioned brains studied. The increase in the number of reactive astrocytes might have been induced by the stimulatory effects of neurotrophic factors, or growth factors, produced around the lesioned site. The constancy in the number of reactive astrocytes after 3 and 4 weeks in the lesioned areas may have been due to the termination of the initial phase of the repair process, i.e. space-filling. Reactive astrocytes which were stained by GFAP-Ab were separated into two groups, based on the presence of CD28, i.e. CD28-positive and CD28-negative reactive astrocytes. The presence of CD28 might confer certain functions via calcium-mediated mechanisms on CD28-positive astrocytes in addition to the constructive role mediated by GFAP.
Animals↗
Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.
Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.
ATP-Binding Cassette Transporters↗
Identification in rat bile of glutathione conjugates of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether, a nephrotoxic degradate of the anesthetic agent sevoflurane.
Recent studies have indicated that the nephrotoxicity of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether ("Compound A"), a breakdown product of the inhaled anesthetic sevoflurane, may be mediated by a reactive intermediate(s) generated via the cysteine conjugate beta-lyase pathway. In order to gain a better understanding of glutathione (GSH)-dependent metabolism of Compound A, the present study was carried out with the primary goal of detecting and characterizing Compound A--GSH conjugates. By means of ionspray LC-MS/MS and NMR spectroscopy, a total of four GSH conjugates ("A1-A4") were identified from the bile of rats dosed intraperitoneally with Compound A. A1 and A2 were identified as two diastereomers of S-[1,1-difluoro-2-(fluoromethoxy)-2-(trifluoromethyl)ethyl]glutath ione, while A3 and A4 were identified as (E)- and (Z)-S-[1-fluoro-2-(fluoromethoxy)-2-(trifluoromethyl)-vinyl]glutat hione, respectively. Quantitative analyses indicated that approximately 29% of the administered dose of Compound A was excreted into the bile in the form of the above GSH conjugates over a period of 6 h. Studies conducted in vitro demonstrated that the reaction of Compound A with GSH was catalyzed by both rat liver cytosolic and microsomal glutathione S-transferases (GST), with the two enzyme systems exhibiting different product selectivities. Formation of these GSH conjugates also occurred nonenzymatically at an appreciable rate. These results indicate that spontaneous and enzyme-mediated conjugation with GSH represents a major pathway of metabolism of Compound A in rats. Conjugation of Compound A with GSH in vivo appeared to be catalyzed preferentially by microsomal rather than cytosolic GST, based on comparison of biliary, microsomal, and cytosolic metabolic profiles. By analogy with other haloalkenes, further metabolism of the corresponding cysteine conjugates of Compound A by renal cysteine conjugate beta-lyase may lead to the formation of reactive acylating agents, which would be expected to bind covalently to cellular macromolecules and cause organ-selective nephrotoxicity.
Animals↗
Molecular epidemiology of measles virus: identification of pathways of transmission and implications for measles elimination.
The nucleotide sequences of either the hemagglutinin or nucleoprotein genes from wild type measles viruses isolated in the United States between 1989 and 1992 differed by < 0.5%. This suggests that the majority of viruses associated with resurgence of measles in the United States belonged to a single indigenous genotype. In contrast, wild type viruses isolated from sporadic outbreaks of measles in the United States during 1994 were genetically heterogeneous. These viruses were more closely related to wild type viruses previously circulating in Europe, Africa, or Japan and were epidemiologically linked to importations or no known source. In addition to demonstrating the utility of genetic analysis in understanding the epidemiology of measles, these data suggest that the transmission of the indigenous virus was interrupted after the 1989-1992 epidemic. Measures to further reduce the incidence of measles in the United States should include efforts to control importation and subsequent spread of measles.
Base Sequence↗
IL-10 selectively regulates murine Ig isotype switching.
A role for IL-10 in regulating Ig isotype switching directly at the level of the murine B cell has not been previously reported. In this report we show that IL-10 selectively up-regulated IgM to IgG3 class switching in lipopolysaccharide (LPS)-activated cultures through a direct effect on membrane (m) IgM+IgG3(-)B cells in vitro. IL-10 stimulated a 3- to 4-fold enhancement (from 6-8 to 20-30%) in membrane mIgG3(+) cells and a significant increase in Smu-Sgamma3 DNA rearrangement events as measured by digestion-circularization PCR (DC-PCR) over that observed with LPS alone. IL-10 induction of switching to IgG3 was not accompanied by a corresponding increase in the steady-state levels of germline CHgamma3 RNA. By contrast, IL-10 strongly inhibited the transforming growth factor-beta-mediated generation of mIgA+ cells and Smu-Salpha DNA rearrangement events in LPS-, but not CD40 ligand (CD40L)-activated B cells. This effect was not accompanied by changes in the steady-state levels of germline CHalpha RNA. IL-10 had no effect on IL-4-mediated switching to either IgG1 or IgE in either LPS- or CD40L-activated B cells. Thus, IL-10 can either enhance or suppress switching to particular murine Ig isotypes but it differs from most other murine cytokines in that its effects on switching do not appear to be associated with changes in the corresponding steady-state levels of germline CH RNA.
Animals↗
IFN-gamma is a potent inducer of Ig secretion by sort-purified murine B cells activated through the mIg, but not the CD40, signaling pathway.
IFN-gamma has been shown to either stimulate or inhibit Ig secretion. No studies have yet addressed the basis for these seemingly conflicting properties nor whether IFN-gamma acted directly at the level of the B cell to mediate its effects. Thus, we studied the ability of IFN-gamma to regulate Ig secretion in sort-purified, resting murine B cells that were >99% Ig+, activated either through membrane Ig using unconjugated or dextran-conjugated anti-IgD antibodies (alphadelta-dex) or through CD40 using soluble or membrane CD40 ligand (CD40L). B cells activated with alphadelta-dex proliferated but do not secrete Ig, even in the presence of IL-1 + IL-2. We demonstrate that IFN-gamma only when added subsequent to B cell stimulation with alphadelta-dex, but not unconjugated anti-IfD antibody, plus IL-1 + IL-2 induces up to 100-fold enhancements in Ig secretion and in the numbers of Ig-secreting cells. The predominant Ig isotype secreted is IgM, with IgG3 and IgG2a comprising the majority of non-IgM antibody. IFN-gamma must act in concert with IL-2 for stimulation of Ig secretion. Further, IFN-gamma synergizes with IL-3 + granulocyte-macrophage colony stimulating factor for induction of Ig synthesis. IFN-gamma also enhances IgA syntheses by transforming growth factor-beta-induced membrane IgA+ cells. By contrast, 125IIFN-gamma fails to stimulate Ig secretion in B cells activated with CD40L in the presence or absence of IL-1 + IL-2 or IL-4. However, the combination of CD40L and alphabeta-dex is strongly synergistic for IFN-gamma-induced Ig secretion. Thus, these data establish that IFN-gamma can act directly on the B cell to induce Ig synthesis without the participation of any other cell and demonstrates that the mode of activation of the B cell plays an important role in directing the action of IFN-gamma.
Animals↗
Microwave treatment enhances detection of RNA and DNA by in situ hybridization.
The use of oligonucleotide probes with nonisotopic detection systems has made in situ hybridization (ISH) more accessible for use in diagnostic pathology; however, ISH using formalin-fixed, paraffin-embedded tissues remains much more sensitive when performed with radioactively labeled probes compared with nonisotopic reporter systems, especially using oligonucleotide probes. We investigated the effects of microwave pretreatment on the detection of RNA and DNA in formalin-fixed, paraffin-embedded tissues in an attempt to improve the sensitivity of ISH with digoxigenin-labeled probes. Titration of normal tissues with oligonucleotide probe cocktails for albumin, prolactin, chromogranin (Cg) A and B mRNAs and a cDNA probe for JC virus showed a significant increase in sensitivity with the use of microwave treatment step rather than heating at 70 degrees C in 2 x SSC for 30 min. Analysis of various solutions used for microwaving showed that 10 mM citrate buffer was more effective than 2 x SSC, phosphate-buffered saline, tissue unmasking fluid, or water. A 15- to 20-min period of microwaving in an 800-W oven produced optimum results. Analysis of a group of tumors for albumin and CgA and B mRNAs and infected brain biopsies for JC virus DNA showed increased sensitivity of the microwave technique using digoxigenin-labeled probes. These results show that microwave treatment can enhance the detection of mRNA and DNA in formalin-fixed, paraffin-embedded tissues.
DNA↗
Immunogenicity and antigenicity of the ATPase/helicase domain of the hepatitis C virus non-structural 3 protein.
The immunogenicity and antigenicity of an enzymatically functional (ATPase/helicase) recombinant protein encompassing residues 1207-1612 of the hepatitis C virus (HCV) non-structural 3 (NS3) protein was characterized using B10 congenic mice. Previous studies have indicated a high frequency of NS3-specific antibodies in HCV-infected humans. Similarly, all six immunized murine haplotypes were antibody responders to the NS3 ATPase/helicase domain, with the H-2k and H-2s haplotypes as high responders. As also observed in HCV-infected humans, the murine NS3 antibodies were predominantly directed to conformational determinants. Irrespective of the murine haplotype, IgG1 predominated in the primary anti-NS3 response, whereas IgG1 and IgG2b predominated in the secondary response. The antibody responder hierarchy was reiterated at the T cell level, with the H-2k and the H-2s haplotypes as the best responders. In both the H-2d and H-2k haplotypes ATPase/helicase-primed T cells secreted interleukin 2 and interferon gamma, corroborating observations from HCV-infected humans. In the H-2d, H-2k and H-2s haplotypes the fine specificity of the T cell recognition of the ATPase/helicase domain was further characterized. Multiple, although generally weak, T cell recognition sites were found for all three haplotypes. The large size of the NS3 protein together with the presence of multiple class II binding motifs explain the high prevalence of NS3 antibodies in immunized mice and predict a similar explanation for the observed high frequency of NS3-specific antibodies in HCV-infected humans.
Adenosine Triphosphatases↗
Dispersion of human Y chromosome haplotypes based on five microsatellites in global populations.
We have analyzed five microsatellite loci from the nonrecombining portion of the human Y chromosome in 15 diverse human populations to evaluate their usefulness in the reconstruction of human evolution and early male migrations. The results show that, in general, most populations have the same set of the most frequent alleles at these loci. Hypothetical ancestral haplotypes, reconstructed on the basis of these alleles and their close derivatives, are shared by multiple populations across racial and geographical boundaries. A network of the observed haplotypes is characterized by a lack of clustering of geographically proximal populations. In spite of this, few distinct clusters of closely related populations emerged in the network, which are associated with population-specific alleles. A tree based on allele frequencies also shows similar results. Lack of haplotypic structure associated with the presumed ancestral haplotypes consisting of individuals from almost all populations indicate a recent common ancestry and/or extensive male migration during human evolutionary history. The convergent nature of microsatellite mutation confounds population relationships. Optimum resolution of Y chromosome evolution will require the use of additional microsatellite loci and diallelic genetic markers with lower mutation rates.
Alleles↗
Transforming growth factor-beta and p27 expression in pituitary cells.
Transforming growth factor-beta (TGF beta) is a member of a family of growth factors that regulates differentiation and cellular proliferation in a wide variety of tissues, including the anterior pituitary gland. TGF beta regulates the expression of various proteins, including p27Kipl (p27), a cell cycle inhibitory protein. The cell types in normal rat anterior pituitary producing TGF beta1, one of the principal isoforms of TGF beta, and p27 were examined by in situ methods. The regulation of p27 messenger RNA (mRNA) and protein by TGF beta1 was also examined in cultured anterior pituitary cells. In situ hybridization, in situ reverse transcriptase PCR, and immunocytochemical staining for pituitary hormones showed that PRL, TSH, and gonadotroph cells all had a higher percentage of cells expressing TGF beta1 mRNA and p27 protein than did GH and ACTH cells. After treatment with 10(-9) M TGF beta1 in vitro for 3 days, there were significant decreases in p27 mRNA and protein levels (P < 0.05) in normal pituitary cells. The GH3 and GHRH-CL1 cell lines, which secrete PRL and GH, had undetectable p27 protein by immunocytochemical staining and immunoblotting, although the GH3 cell line had p27 mRNA detected by reverse transcriptase PCR. Analysis of [3H]thymidine uptake in cultured dissociated pituitary cells by double staining for hormones showed that only PRL cells had significant proliferative activity during a 3-day cell culture period. There was a biphasic effect of TGF beta1 on PRL cell proliferation, with marked inhibition by 10(-9) M and a slight stimulation by 10(-13) M. These results indicate that there is a differential distribution of both TGF beta1 and p27 in various anterior pituitary cell types and that TGF beta1 directly down-regulates p27 in cultured anterior pituitary cells.
Animals↗
Pancreastatin secretion by pituitary adenomas and regulation of chromogranin B mRNA expression.
Pancreastatin, a carboxyl-terminal amidated peptide derived from chromogranin (Cg)A, inhibits secretion of insulin and parathyroid hormone. Our recent studies found significant amounts of immunoreactive pancreastatin in all pituitary adenomas except prolactin adenomas. To analyze the effects of pancreastatin on pituitary cell function, 17 cultured pituitary adenomas were examined for immunoreactive pancreastatin and pancreastatin secretion by the tumors. The effects of pancreastatin on pituitary hormone secretion and on pituitary hormone (follicle-stimulating hormone and prolactin), CgA, and CgB mRNA levels were also examined. Immunoreactive pancreastatin and CgA were present diffusely in gonadotroph and null cell adenomas, but only a few prolactin adenoma cells expressed pancreastatin or CgA. When cells were treated with hypothalamic peptides, gonadotroph adenomas were the only group that released increased amounts of pancreastatin in response to gonadotropin-releasing hormone (10(-7) mol/L). Pancreastatin (10(-7) mol/L) treatment did not stimulate pituitary hormone secretion significantly. In situ hybridization analyses showed that gonadotropin-releasing hormone and pancreastatin treatment led to significant increases in CgB and follicle-stimulating hormone mRNAs in gonadotroph adenomas, whereas CgA mRNA levels did not change significantly. These results show that there is a differential distribution of pancreastatin secretion in pituitary adenomas and that the hypothalamic hormone gonadotropin-releasing hormone and the CgA-derived peptide pancreastatin can regulate CgB mRNA in gonadotroph adenomas, suggesting an autocrine effect of pancreastatin on pituitary tumor function.
Adenoma↗
Scatter factor binds to thrombospondin and other extracellular matrix components.
Scatter factor (SF) is an angiogenic growth factor that stimulates motility and invasion of carcinoma cells. SF is present in the extracellular matrix (ECM) of breast cancers, where it might act to promote tumor cell invasion and angiogenesis. To investigate how SF is incorporated into the ECM, we studied the binding of SF to various ECM components using a solid-phase binding assay based on the SF enzyme-linked immunosorbent assay. We found that SF binds to a variety of ECM molecules, with different binding capacities. The highest SF binding capacities were observed for thrombospondin-1 (TSP-1), fibronectin (Fn), and heparan sulfate proteoglycan, although SF did not bind to albumin. Mature two-chain SF and precursor single-chain SF bound approximately equally well to TSP-1 and Fn. Moreover, two SF alpha-chain peptides (NK1 and NK2) both bound to TSP-1 and Fn, suggesting that the whole SF molecule is not required for binding. Based on binding competition assays, TSP-1 exhibited higher affinity for SF than did nine other ECM molecules, including Fn and heparan sulfate proteoglycan. Although heparin in solution potently inhibited the binding of SF to TSP-1-coated surfaces, even very high concentrations of heparin could not elute SF already bound to TSP-1. SF binding was modulated by binding interactions among ECM molecules (TSP-1-Fn, TSP-1-collagen I, and Fn-collagen I), suggesting that the matrix capacity to bind SF depends upon its exact composition. SF bound in a dose-dependent fashion to ECMs secreted by three human breast carcinoma cell lines. Binding of SF to matrices from all three cell lines was significantly inhibited by preincubation of the matrices with antibodies against TSP-1, whereas antibodies against several other ECM components were less effective or ineffective in inhibiting SF binding. In addition, TSP-1 markedly inhibited chemotaxis of microvascular endothelial cells toward SF and SF-induced angiogenesis in the rat cornea neovascularization assay. Our findings suggest that 1) SF interacts with a variety of ECM components, 2) high affinity SF-TSP-1 interactions may mediate the binding of SF to the breast cancer matrix, and 3) the SF-TSP-1 interaction may contribute to modulation of angiogenesis. Possible implications of these findings for tumor angiogenesis are discussed.
Animals↗
Immunohistochemical localization of metallothionein in human prostate cancer.
PURPOSE: We investigated the expression of metallothionein in human prostate cancer. MATERIALS AND METHODS: Formalin fixed and paraffin embedded tissue specimens from 45 patients with primary prostate cancer were stained for metallothionein using a standard immunohistochemical technique. RESULTS: Metallothionein was detected in 15 of 45 prostate cancers (33.3%). Cytoplasmic and nuclear staining occurred in most cells. Additionally, metallothionein was found in basement membrane surrounding the cancer cells in 2 cases with metallothionein expression, and in secretory products of the lumen in a few cases. Statistical analysis for metallothionein expression related to tumor grade revealed a significant difference between high (7 to 10) and low (2 to 4) Gleason scores (p < 0.001), as well as between middle (5 and 6) and low scores (p < 0.05). However, no relationship was found with clinical stage. CONCLUSIONS: Our data suggest a close correlation of metallothionein expression with tumor grade and a wide range of metallothionein expression in prostate cancer.
Aged↗
Scatter factor protein levels in human breast cancers: clinicopathological and biological correlations.
Scatter factor (SF) is an invasogenic and angiogenic cytokine the cellular receptor of which is encoded by a proto-oncogene (c-met). We measured the immunoreactive SF content (nanograms of SF per milligram of protein) in tissue extracts from 166 breast cancers and correlated the values with various known prognostic parameters. Invasive cancers had nearly four times greater SF content than did ductal carcinoma in situ, and the difference was statistically significant (P < 0.02, two-tailed t-test). However, there were no significant differences in SF content among different histological types of invasive cancer. Invasive cancers that had spread to axillary lymph nodes exhibited higher SF content than did invasive cancers without regional spread (P < 0.02), but the difference in SF content between node-positive and node-negative tumors was not as great as that between invasive and ductal carcinoma in situ tumors. There was a trend toward increased SF content in larger primary tumors as compared with smaller tumors, but statistical comparison revealed borderline significance (0.05 < P < 1.0). There was no significant correlation between SF content and other parameters, including estrogen receptor, progesterone receptor, DNA ploidy, S phase, or Scarff-Bloom-Richardson score. We also measured the content of von Willebrand factor (a marker of blood vessels) and interleukin-1 beta (a pro-inflammatory cytokine) in the same tumor extracts. SF content showed a strong positive correlation with von Willebrand factor content (P < 0.001) but did not appear to be correlated with interleukin-1 beta. These findings suggest that SF is correlated with several other clinicopathological indicators of aggressive tumor behavior, consistent with the hypothesis that SF is a biological factor that may play a role in breast cancer pathogenesis.
Biomarkers, Tumor↗
Periodontal repair in dogs: evaluation of rhBMP-2 carriers.
This study evaluated candidate carriers for recombinant human bone morphogenetic protein-2 (rhBMP-2)-driven periodontal regeneration. Previous experiments indicated the ability of rhBMP-2 in a particulate delivery system to result in substantial regeneration of bone and periodontal regeneration. In the present study, canine demineralized bone matrix (DBM), bovine deorganified crystalline bone matrix (Bio-Oss), an absorbable collagen sponge (ACS) of type I bovine collagen, poly(D,L-lactide-co-glycolide) microparticles (PLGA), and polylactic acid granules (Drilac) were tested for their ability to support rhBMP-2 (0.2 mg/mL implant volume)-driven periodontal regeneration. The implants were tested in routine critical size canine supra-alveolar periodontal defects with transgingival tooth positioning. Contralateral defects in six beagle dogs were semirandomly assigned to receive: DBM/rhBMP-2, DBM (no rhBMP-2), Bio-Oss/rhBMP-2, ACS/rhBMP-2, PLGA/rhBMP-2, or Drilac/rhBMP-2. Animals were sacrificed 8 weeks postsurgery, and block sections of the defects were processed for light microscopy. Substantial bone regeneration was observed in all defects implanted with rhBMP-2. Other measures of periodontal healing, including cementum regeneration and presence of ankylosis, were more variable between the implants. DBM and Bio-Oss performed well as carriers for rhBMP-2-driven periodontal regeneration, although other impediments to their clinical use exist. This study indicates that qualities of the carrier system, including its space-maintaining capacity can affect the ability of rhBMP-2 to regenerate both alveolar bone and periodontal attachment.
Alveolar Bone Loss↗