Protective effects of Acetobacter ghanensis against gliadin toxicity in intestinal epithelial cells with immunoregulatory and gluten-digestive properties.

PURPOSE: The aim of this study was to establish whether Acetobacter ghanensis, the probiotic characteristics of which were evaluated previously, attenuates gliadin-induced toxicity in intestinal epithelial cells with gluten-digestive and immunoregulatory properties. METHODS: A co-culture model of human intestinal epithelial cell (Caco-2) monolayers on top of peripheral blood mononuclear cells (PBMCs) obtained from patients with celiac disease (CD) was established. The gluten-digestive properties of A. ghanensis were determined by checking bacterial growth in a medium containing gluten as the main nitrogen source. The mRNA levels of genes encoding TJ-associated proteins were measured by quantitative real-time PCR (qRT-PCR). The concentrations of IL-6 and TNFα were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that PT-gliadin disrupted intestinal barrier integrity by modulating the expression of TJ-associated genes encoding zonulin (increased by ~ 60%), zonula occludens-1 (ZO-1) (decreased by ~ 22%), and occludin (decreased by ~ 28%) in Caco-2 cells. Furthermore, PT-gliadin treatment in Caco-2 cells was associated with increased concentrations of IL-6 (~ 1.6-fold) and TNFα (~ twofold) from PBMCs. These modulatory effects of PT-gliadin, however, were suppressed when Caco-2 cells were subjected to A. ghanensis in the presence of PT-gliadin. As a factor underlying these protective effects, we showed that A. ghanensis could digest gluten peptides. CONCLUSIONS: To our knowledge, the current study is the first to demonstrate that A. ghanensis improves intestinal barrier functions by attenuating the modulatory effects of PT-gliadin with immunoregulatory and gluten-digestive properties.

Copy number increase of oncoprotein CIP2A is associated with poor patient survival in human head and neck squamous cell carcinoma.

BACKGROUND: CIP2A, an inhibitor of PP2A tumour suppressor function, is a widely overexpressed biomarker of aggressive disease and poor therapy response in multiple human cancer types. METHODS: CIP2A and DPPA4 copy number alterations and expression were analysed by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in different cell lines and a tissue microarray of 52 HNSCC patients. Results were correlated with patient survival and other clinicopathological data. RESULTS: CIP2A and DPPA4 copy number increase occurred at a relatively high frequency in human HNSCC patient samples. CIP2A but not DPPA4 FISH status was significantly associated with patient survival. CIP2A detection by combining IHC with FISH yielded superior resolution in the prognostication of HNSCC. CONCLUSIONS: CIP2A copy number increase is associated with poor patient survival in human HNSCC. We suggest that the reliability and prognostic value of CIP2A detection can be improved by performing FISH analysis to CIP2A IHC positive tumours.

A Novel Mutation in the Promoter Region of the ß-Globin Gene: HBB: c.-127G > C.

Novel ß-globin gene mutations are still occasionally being reported, especially when evaluating milder phenotypes. We report here a novel putative mutation in the promoter region of the ß-globin gene and assess its clinical implications. A family, parents and four siblings, with hematological and clinical features suspected of being ß-globin gene mutation(s), were involved in this study. In addition to hematological and clinical evaluations of the whole family, molecular analyses of the ß-globin gene were performed by direct sequencing. Sequencing of the ß-globin gene revealed a novel genomic alteration in the regulatory region of the gene. This novel genomic alteration was defined as HBB: c.-127G > C according to the Human Genome Variation Society (HGVS) nomenclature. Two siblings were found to be carriers of the HBB: c.-127G > C mutation, while the other two siblings were carriers of the codon 8 (-AA) (HBB: c.25_26delAA) deletion of the ß-globin gene. The mother was a compound heterozygote for the codon 8 and HBB: c.-127G > C mutations. Based on hematological and clinical evaluations, we conclude that this novel ß-globin gene promoter region change would be associated with a mild phenotype of ß-thalassemia (ß-thal).

Myxoma Virus Combination Therapy Enhances Lenalidomide and Bortezomib Treatments for Multiple Myeloma.

This study aimed to explore the effectiveness and safety of Myxoma virus (MYXV) in MM cell lines and primary myeloma cells obtained from patients with multiple myeloma. Myeloma cells were isolated from MM patients and cultured. MYXV, lenalidomide, and bortezomib were used in MM cells. The cytotoxicity assay was investigated using WST-1. Apoptosis was assessed through flow cytometry with Annexin V/PI staining and caspase-9 concentrations using ELISA. To explore MYXV entry into MM cells, monoclonal antibodies were used. Moreover, to explore the mechanisms of MYXV entry into MM cells, we examined the level of GFP-labeled MYXV within the cells after blocking with monoclonal antibodies targeting BCMA, CD20, CD28, CD33, CD38, CD56, CD86, CD117, CD138, CD200, and CD307 in MM cells. The study demonstrated the effects of treating Myxoma virus with lenalidomide and bortezomib. The treatment resulted in reduced cell viability and increased caspase-9 expression. Only low-dose CD86 blockade showed a significant difference in MYXV entry into MM cells. The virus caused an increase in the rate of apoptosis in the cells, regardless of whether it was administered alone or in combination with drugs. The groups with the presence of the virus showed higher rates of early apoptosis. The Virus, Virus + Bortezomib, and Virus + Lenalidomide groups had significantly higher rates of early apoptosis (p < 0.001). However, the measurements of late apoptosis and necrosis showed variability. The addition of MYXV resulted in a statistically significant increase in early apoptosis in both newly diagnosed and refractory MM patients. Our results highlight that patient-based therapy should also be considered for the effective management of MM.

Prevalence and significance of the MEFV gene mutations in childhood Henoch-Schönlein purpura without FMF symptoms.

Familial Mediterranean fever (FMF) has been reported more frequently in patients presenting with Henoch-Schönlein purpura (HSP) than in the general population. But, there is no clear knowledge about MEFV mutations in patients with HSP. We investigated the prevalence of MEFV mutations in children with HSP and without FMF whether these mutations have any effect on the disease course or complications. A total of 76 children with HSP who had no typical symptoms of FMF were screened for the mutations in exon 2 and exon 10 of the MEFV gene. Eleven of 76 patients (14.4 %) were heterozygous (E148Q in 5, M694V in 4, M680I in 1, E148V in 1), 5 (6.6 %) were homozygous (M694V/M694V in 4, V726A/V726A in 1), and 2 (2.6 %) were compound heterozygous (E148Q/M694V mutations in 1 and L110P/E148Q mutations in 1). Altogether, 7 patients carried 2 mutated MEFV alleles (9.2 %), which was higher than that observed in the general Turkish population (1 %). No significant differences in joint, gastrointestinal, renal involvement, or subcutaneous edema, and also acute phase reactants including leukocyte count, erythrocyte sedimentation rate, and serum C-reactive protein concentration were found between the groups. The prevalence of the two allele-MEFV mutations in patients with HSP was found higher than that of the general population. However, it seems that MEFV gene mutations may not have any effect on the clinical presentation of HSP.

Alloreactive memory B cell detection by flow cytometric cross match using polyclonally activated memory B cell culture supernatants.

In addition to alloantibodies, alloreactive memory B cell (mBC) evaluation has a potential for immunological risk assessment during transplantation processes. For the alloreactive mBCs evaluation currently, direct Flow Cytometric (FC) analysis using the HLA tetramer staining is an option. Evaluation of alloantibodies produced by the polyclonally stimulated alloreactive mBCs in in vitro culture system seems to be another useful approach, but this needs further downstream applications. In this study, we investigated the usefulness of the Flow Cytometric Cross Match (FCXM-supernatant) in which in vitro polyclonally activated mBCs culture supernatants and potential donor's lymphocytes being used for the mBC detection. FCXM-supernatant assays were performed between culture supernatants of polyclonally activated mBCs obtained from 4 allosensitized multiparous women and 14 renal transplant patients, and their non-alloimmunized spouses' or donors' lymphocytes, and vice versa. HLA typing was performed by SSP method. Anti-HLA antibodies produced by in vitro activated alloreactive mBCs were also evaluated by the Luminex assays. The success of in vitro polyclonal activation of mBCs was evaluated by a total IgG ELISA test and antibody secreting cell analyses by FC. Donor specific alloreactive mBCs were detected by FCXM-supernatant in 45% of the 18 allosensitized cases. Detection rate was 85% (6 out of 7) in the strongly allosensitized cases. No alloreactive mBCs was detected in control cases without allosensitization. FCXM-supernatant negative results of the allosensitized cases were related to low level of allosensitization and insufficient polyclonal stimulation evaluated by total IgG antibody tests of the supernatants. We herein report a practical methodology for alloreactive mBC detection as a donor specific manner using the FCXM-supernatant assay so that this would easily be transformed into a routine test performed in tissue typing laboratories.

Effects of idiopathic erythrocytosis on the left ventricular diastolic functions and the spectrum of genetic mutations: A case control study.

BACKGROUND: We have aimed at exposing left ventricular diastolic functions and the presence of known genetic mutations for familial erythrocytosis, in patients who exhibit idiopathic erythrocytosis. METHODS: Sixty-four patients with idiopathic erythrocytosis (mean age, 46.4 ± 2.7 years) and 30 age-matched healthy subjects were prospectively evaluated. The regions of interest of the erythropoietin receptor, hemoglobin beta-globin, von Hippel-Lindau, hypoxia-inducible factor 2 alpha, and Egl-9 family hypoxia-inducible factor genes were amplified by PCR. Left ventricular (LV) mass was measured by M-mode and 2-dimensional echocardiography. LV diastolic functions were assessed by conventional echocardiography and tissue Doppler imaging. RESULTS: As a result of genetic analyses, genetic mutations for familial erythrocytosis were detected in 5 patients. It has been observed in our study that the risk of cardiovascular disorders is higher in patients. Interventricular septum thickness, left atrial diameter, and some diastolic function parameters such as deceleration time and isovolumetric relaxation time have been found to be significantly higher in idiopathic erythrocytosis group than in the controls. CONCLUSION: This study has shown that LV diastolic functions were impaired in patients with idiopathic erythrocytosis. In this patient group with increased risk of cardiovascular disorders, the frequent genetic mutations have been detected in 5 patients only. Therefore, further clinical investigations are needed as novel genetic mutations may be discovered in patients with idiopathic erythrocytosis because of cardiovascular risk.

The association between intragenic SNP haplotypes and mutations of the beta globin gene in a Turkish population.

Identification of the beta globin gene mutation-related haplotypes is of interest for the delineation of the clinical heterogeneity as well as understanding of the origin and spreading of the beta globin gene mutations. We screened the whole beta globin gene in 197 Turkish patients by direct sequencing and performed Haploview analyses for beta globin gene haplotyping using five common intragenic SNPs; rs713040, rs10768683, rs7480526, rs7946748, and rs1609812. We found 25 different beta globin gene point mutations by sequencing. A Turkish type of inv/del mutation by MLPA and Gap-PCR was also detected with additional studies. The seven most common mutations with higher frequency of 5% were IVS-I-110 (G>A) (35.6%), Hb S(10.6%), IVS-I-6 (T>C) (7.4%), IVS-I-1 (G>A) (6.9%), IVS-II-1 (G>A) (6.9%), Cod8(-AA) (6%), IVS-II-745 (C>G) (5.1%) and accounted for 78.7% of all mutations. We identified seven different haplotypes (Haplotype I-VII) using five intragenic single nucleotide polymorphisms (SNPs) genotyped by sequencing of the beta globin gene. The association between the mutations and the haplotypes was defined for 16 different mutations. We suggest that haplotyping by these five intragenic SNPs will provide useful information about the origin of the mutations and gene flow among as well as the explanation of the clinical heterogeneity.

The association between inherited thrombophilias and pregnancy-related hypertension recurrence.

PURPOSE: The aim of this study was to determine the association between inherited thrombophilias and pregnancy-related hypertension recurrence. METHODS: In this case-control study, blood samples were obtained from patients who had at least two pregnancies complicated with pregnancy-related hypertension (n = 41) and healthy, normotensive pregnancies delivered without any complication (n = 38). Following the DNA extraction, samples were tested for factor V Leiden, prothrombin G20210A and homozygous methylene tetrahydrofolate reductase (MTHFR) C677T mutations using reverse hybridization method. RESULTS: Common inherited thrombophilias were present in 26.8% of women with recurrent pregnancy-related hypertension group and 23.7% of control subjects (odds ratio 1.1; 95% confidence interval, 0.6-1.9). No significant difference was observed between two groups in terms of factor V Leiden, prothrombin G20210A and MTHFR C677T mutations. CONCLUSION: Our data suggest that factor V Leiden, prothrombin G20210A and MTHFR C677T mutations are not associated with pregnancy-related hypertension recurrence.

The effect of HBB: c.*+96T>C (3'UTR +1570 T>C) on the mild b-thalassemia intermedia phenotype.

Hemoglobin beta (HBB): c.*+96T>C substitution is very rare among ß-globin gene mutations and its clinical significance remains to be clarified. The present study aimed to investigate the role of HBB: c.*+96T>C in the ß-thalassemia intermedia phenotype in a Turkish family. The proband and parents were screened for ß-globin gene mutations via direct sequencing. Hematological and physical examination results were recorded, and correlated according to genotype. The proband was compound heterozygous for Cod 8 (-AA) and HBB: c.*+96T>C, whereas his mother and father were heterozygous for Cod 8 (-AA) and HBB: c.*+96T>C, respectively. The father had almost normal hematological findings, whereas the mother had the typical ß-thalassemia trait phenotype. The proband was diagnosed as mild ß-thalassemia intermedia based on hepatosplenomegaly and hematological findings. To the best of our knowledge this is the first report of HBB: c.*+96T>C mutation in a Turkish family. HBB: c.* 96T>C substitution is a very rare, but clinically relevant ß-globin gene mutation. Additionally, we think that if 1 spouse is a carrier for ß-globin gene mutation the other should be screened for silent mutations, such as HBB: c.*+96T>C mutation of the ß-globin gene, even if she/he does not have any clinical or hematological signs of the ß-thalassemia trait phenotype.

Insertion/deletion polymorphism and serum activity of the angiotensin-converting enzyme in Turkish patients with obstructive sleep apnea syndrome.

This study determined the allelic frequency and genotypic distribution of an angiotensin-converting enzyme (ACE) polymorphism and serum ACE activity in Turkish patients with obstructive sleep apnea syndrome (OSAS). A colorimetric assay measured serum ACE activity in 73 of 97 subjects. Frequencies for II, ID, and DD genotypes were 19.6, 53.6, and 26.8% in the OSAS group and 15, 38, and 47% in the control group, respectively (P = 0.02). The I allele frequency was higher in the OSAS group than in the healthy control group (P = 0.02). Carrying the I allele (II or ID genotypes) increased OSAS risk 2.41 times in the Turkish population. Mean ACE activity was significantly lower in patients with the II genotype than in the DD genotype (P = 0.011), and ACE activity was significantly lower in patients with severe OSAS than in those with mild OSAS (P = 0.006). Our results suggest that II and ID genotypes of the ACE gene increase the risk of developing OSAS in the Turkish population.

Comparison of Cytotoxic Flow Cytometric Cross Match With Complement Dependent Lymphocytotoxicity and Flow Cytometric Cross Match in Renal Transplant Patients.

Cytotoxic flow cytometric crossmatch (cFCXM), identified by detecting complement-mediated cytotoxic cell death in addition to the capability of showing the alloantibodies binding onto lymphocytes at the same time, can reduce the necessary time and workload in evaluating alloantibodies. More data from clinical samples are needed for cFCXM to be accepted by tissue typing laboratories. In this study, we compared cFCXM with complement-dependent lymphocytotoxicity and standard flow cytometric crossmatch in 41 renal pretransplant patients. A comparison of the obtained data was performed using Spearman's correlation test. We found that cFCXM showed no statistically significant differences with complement-dependent lymphocytotoxicity and flow cytometric crossmatch. We believe that cFCXM can be used in clinical laboratories in the near future following intra-laboratory validation.

Gap-PCR Screening for Common Large Deletional Mutations of ß-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/Deletion (δß)0 Mutation in Antalya.

OBJECTIVE: Although the calculated carrier frequency for point mutations of the ß-globin gene is around 10% for Antalya Province, nothing is known about the profile of large deletional mutations involving the ß-globin gene. In this study, we aimed to screen common deletional mutations in the ß-globin gene cluster in patients for whom direct DNA sequencing was not able to demonstrate the mutation(s) responsible for the disease phenotype. MATERIALS AND METHODS: Thirty-one index cases selected with a series of selection events among 60 cases without detected ß-globin gene mutation from 580 thalassemia-related cases tested by direct sequencing over the last 4 years in our diagnostic center were screened for the most common 8 different large deletional mutations of the ß-globin gene cluster by gap-PCR. RESULTS: We detected 1 homozygous and 9 heterozygous novel unrelated cases for the Turkish inversion/deletion (δß)0 mutation in our series of 31 cases. Our study showed that the Turkish inversion/deletion (δß)0 mutation per se accounts for 16.6% of the unidentified causative alleles and also accounts for 1.5% of all detected mutations over the last 4 years in our laboratory. CONCLUSION: Since molecular diagnosis of deletional mutations in the ß-globin gene cluster warrants different approaches, it deserves special attention in order to provide prenatal diagnosis and prevention opportunities to the families involved. We conclude that the Turkish inversion/deletion (δß)0, as the most prevalent deletional mutation detected so far, has to be routinely tested for in Antalya, and the gap-PCR approach has valuable diagnostic potential in the patients at risk.

Chk1 targeting reactivates PP2A tumor suppressor activity in cancer cells.

Checkpoint kinase Chk1 is constitutively active in many cancer cell types and new generation Chk1 inhibitors show marked antitumor activity as single agents. Here we present a hitherto unrecognized mechanism that contributes to the response of cancer cells to Chk1-targeted therapy. Inhibiting chronic Chk1 activity in cancer cells induced the tumor suppressor activity of protein phosphatase protein phosphatase 2A (PP2A), which by dephosphorylating MYC serine 62, inhibited MYC activity and impaired cancer cell survival. Mechanistic investigations revealed that Chk1 inhibition activated PP2A by decreasing the transcription of cancerous inhibitor of PP2A (CIP2A), a chief inhibitor of PP2A activity. Inhibition of cancer cell clonogenicity by Chk1 inhibition could be rescued in vitro either by exogenous expression of CIP2A or by blocking the CIP2A-regulated PP2A complex. Chk1-mediated CIP2A regulation was extended in tumor models dependent on either Chk1 or CIP2A. The clinical relevance of CIP2A as a Chk1 effector protein was validated in several human cancer types, including neuroblastoma, where CIP2A was identified as an NMYC-independent prognostic factor. Because the Chk1-CIP2A-PP2A pathway is driven by DNA-PK activity, functioning regardless of p53 or ATM/ATR status, our results offer explanative power for understanding how Chk1 inhibitors mediate single-agent anticancer efficacy. Furthermore, they define CIP2A-PP2A status in cancer cells as a pharmacodynamic marker for their response to Chk1-targeted therapy.

ETS1 mediates MEK1/2-dependent overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A) in human cancer cells.

EGFR-MEK-ERK signaling pathway has an established role in promoting malignant growth and disease progression in human cancers. Therefore identification of transcriptional targets mediating the oncogenic effects of the EGFR-MEK-ERK pathway would be highly relevant. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently characterized human oncoprotein. CIP2A promotes malignant cell growth and is over expressed at high frequency (40-80%) in most of the human cancer types. However, the mechanisms inducing its expression in cancer still remain largely unexplored. Here we present systematic analysis of contribution of potential gene regulatory mechanisms for high CIP2A expression in cancer. Our data shows that evolutionary conserved CpG islands at the proximal CIP2A promoter are not methylated both in normal and cancer cells. Furthermore, sequencing of the active CIP2A promoter region from altogether seven normal and malignant cell types did not reveal any sequence alterations that would increase CIP2A expression specifically in cancer cells. However, treatment of cancer cells with various signaling pathway inhibitors revealed that CIP2A mRNA expression was sensitive to inhibition of EGFR activity as well as inhibition or activation of MEK-ERK pathway. Moreover, MEK1/2-specific siRNAs decreased CIP2A protein expression. Series of CIP2A promoter-luciferase constructs were created to identify proximal -27 to -107 promoter region responsible for MEK-dependent stimulation of CIP2A expression. Additional mutagenesis and chromatin immunoprecipitation experiments revealed ETS1 as the transcription factor mediating stimulation of CIP2A expression through EGFR-MEK pathway. Thus, ETS1 is probably mediating high CIP2A expression in human cancers with increased EGFR-MEK1/2-ERK pathway activity. These results also suggest that in addition to its established role in invasion and angiogenesis, ETS1 may support malignant cellular growth via regulation of CIP2A expression and protein phosphatase 2A inhibition.

The effect of CYP1A2 gene polymorphisms on Theophylline metabolism and chronic obstructive pulmonary disease in Turkish patients.

Cytochrome P450 (CYP) 1A2 gene polymorphisms are thought to be involved in the metabolism of theophylline (TP). We aimed to investigate the effect of CYP1A2*1C, CYP1A2*1D, CYP1A2*1E, and CYP1A2*1F polymorphisms of the CYP1A2 on TP metabolism by PCR-RFLP in 100 Turkish patients with chronic obstructive pulmonary disease (COPD) receiving TP. One hundred and one healthy volunteers were included as control group. The genotype frequencies of the CYP1A2*1D and CYP1A2*1F were found to be significantly different in the patients compared to the controls. The "T" allele at -2467 delT and the "C" allele at -163 C > A in the CYP1A2 displayed association with a significantly increased risk for COPD. "T" allele at - 2467 delT was also associated with a high risk of disease severity in COPD. In conclusion, our data suggest that genetic alterations in CYP1A2 may play a role both in the pharmacogenetics of TP and in the development of COPD.

No association between bone mineral density and transforming growth factor beta gene T(861-20)-C polymorphism in Turkish postmenopausal women.

The aim of the study was to examine whether the TGF-beta1 T(861-20)-C gene polymorphism might be useful in identifying individuals with increased susceptibility to postmenopausal bone loss within the Turkish women population. T(861-20)-C polymorphism was genotyped in 616 postmenopausal women selected from the Turkish population: 311 postmenopausal osteoporotic women (OP) aged 45-65 years (mean age 58 years) and a control group (CG) of 305 postmenopausal women in the same age range (mean age 53 years) with normal bone mineral density. We have not found any significant differences in the frequency of the individual genotypes between the osteoporotic and control groups. The distribution of the T(861-20)-C genotypes was for Lumbar spine, CC, 74.0% in OP, 75.1% in CG; TC, 24.1% in OP, 23.9% in CG; TT, 1.9% in OP, 1.0% in CG; and for femoral neck, CC, 76.8% in OP, 72.8% in CG; TC, 22.1% in OP, 25.5% in CG; TT 1.1% in OP, 1.7% in CG. T(861-20)-C polymorphism was not found to be associated with bone mineral density in postmenopausal Turkish women. It was argued that this will be a pioneering study for the future research and therapies.

Effects of hormone replacement therapy on bone mineral density in Turkish patients with or without COL1A1 Sp1 binding site polymorphism.

AIM: To evaluate the effects of hormone replacement therapy (HRT) on bone mineral density (BMD) in patients with or without COL1A1 Sp1 binding site polymorphism. METHODS: Non-smoking otherwise healthy postmenopausal women (n=111), who had not received any kind of HRT for at least 3 years (between 2002 and 2005) at the onset of menopause, were included. All patients received 0.625 mg conjugated estrogen/2.5 mg medroxyprogesterone for 18 months. BMD by dual X-ray absorptiometry was measured at the lumbar spine and the femur neck initially and after 18th months of treatment. COL1A1 Sp1 binding site polymorphism was studied using the PCR-RFLP method. RESULTS: After having the results of COL1A1 Sp1 binding site polymorphism, 79 (71.2%) patients were SS, 30(27.0%) were Ss and two (1.8%) were homozygous for ss. The mean age, weight and length of menopausal period were similar between the SS and Ss patients. The Ss heterozygotes had lower BMD values both at the lumbar spine and at the femur neck compared with the SS patients. This difference was also reflected in post treatment measurements. The increase in BMD scores was higher in the SS homozygotes than in the Ss patients. CONCLUSION: Our preliminary data supports the fact that HRT had a lower increase in BMD scores following 18 months of treatment in COL1A1 s allele individuals compared with normal SS individuals. Therefore our study may provide evidence that the Sp1 polymorphism may ameliorate the effects of HRT on BMD, suggesting some additional regimens may be used to support bone strength and to decrease osteoporotic fractures.