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Background: The COVID-19 pandemic reached Germany in spring 2020. No proven treatment for SARS-CoV-2 was available at that time, especially for severe COVID-19-induced ARDS. We determined whether the infusion of mesenchymal stromal cells (MSCs) would help to improve pulmonary function and overall outcome in patients with severe COVID-19 ARDS. We offered MSC infusion as an extended indication to all critically ill COVID-19 patients with a Horovitz index Result: Four out of 5 MSC-treated patients compared to 50% of control patients (9 out of 18) received ECMO support (80%). The MSC group showed a higher Murray score on admission than control patients, reflecting more severe pulmonary compromise (3.5 ± 0.2 versus 2.8 ± 0.3). MSC infusion was safe and well tolerated. The MSC group had a significantly higher Horovitz score on discharge than the control group. Compared to controls, patients with MSC treatment showed a significantly lower Murray score upon discharge than controls. In the MSC group, 4 out of 5 patients (80%) survived to discharge and exhibited good pulmonary function, whereas only 8 out of 18 patients (45%) in the control group survived to discharge. Conclusion: MSC infusion is a safe treatment for COVID-19 ARDS that improves pulmonary function and overall outcome in this patient population.

Targeting metabolism through bioactive key metabolites is an upcoming future therapeutic strategy. We questioned how modifying intracellular lipid metabolism could be a possible means for alleviating inflammation. Using a recently developed chemical probe (SH42), we inhibited distal cholesterol biosynthesis through selective inhibition of Δ(24)-dehydrocholesterol reductase (DHCR24). Inhibition of DHCR24 led to an antiinflammatory/proresolving phenotype in a murine peritonitis model. Subsequently, we investigated several omics layers in order to link our phenotypic observations with key metabolic alterations. Lipidomic analysis revealed a significant increase in endogenous polyunsaturated fatty acid (PUFA) biosynthesis. These data integrated with gene expression analysis, revealing increased expression of the desaturase Fads6 and the key proresolving enzyme Alox-12/15. Protein array analysis, as well as immune cell phenotype and functional analysis, substantiated these results confirming the antiinflammatory/proresolving phenotype. Ultimately, lipid mediator (LM) analysis revealed the increased production of bioactive lipids, channeling the observed metabolic alterations into a key class of metabolites known for their capacity to change the inflammatory phenotype.

The bidirectional communication between the immune and nervous system is important in regulating immune responses. Here we show that the adrenergic nerves of sympathetic nervous system orchestrate inflammation resolution and regenerative programs by modulating repulsive guidance molecule A (RGM-A). In murine peritonitis, adrenergic nerves and RGM-A show bidirectional activation by stimulating the mutual expression and exhibit a higher potency for the cessation of neutrophil infiltration; this reduction is accompanied by increased pro-resolving monocyte or macrophage recruitment, polymorphonucleocyte clearance and specialized pro-resolving lipid mediators production at sites of injury. Chemical sympathectomy results in hyperinflammation and ineffective resolution in mice, while RGM-A treatments reverse these phenotypes. Signalling network analyses imply that RGM-A and β2AR agonist regulate monocyte activation by suppressing NF-κB activity but activating RICTOR and PI3K/AKT signalling. Our results thus illustrate the function of sympathetic nervous system and RGM-A in regulating resolution and tissue repair in a murine acute peritonitis model., Diverse interactions between the nervous and immune systems have been shown, but specific mechanistic insights are still lacking. Here the authors show, using both mouse inflammation models and clinical correlation, that adrenergic nerve may ameliorate inflammation by inducing repulsive guidance molecule A signalling.

Critical conditions such as sepsis following infection or traumatic injury disturb the complex state of homeostasis that may lead to uncontrolled inflammation resulting in organ failure, shock and death. They are associated with endogenous mediators that control the onset of acute inflammatory response, but the central problem remains the complete resolution of inflammation. Omega-3 enriched lipid emulsions (Ω-3+ LEs) were used in experimental studies and clinical trials to establish homeostasis, yet with little understanding about their role on the resolution of inflammation and tissue regeneration. Here, we demonstrate that Ω-3 lipid emulsions (LEs) orchestrate inflammation-resolution/regeneration mechanism during sterile peritonitis and murine polymicrobial sepsis. Ω-3+ LEs recessed neutrophil infiltration, reduced pro-inflammatory mediators, reduced the classical monocyte and enhanced the non-classical monocytes/macrophages recruitment and finally increased the efferocytosis in sepsis. The actions of Ω-3+ LE were 5-lipoxygenase (5-LOX) and 12/15-lipoxygenase (12/15-LOX) dependent. Ω-3+ LEs shortened the resolution interval by 56%, stimulated the endogenous biosynthesis of resolution mediators lipoxin A4, protectin DX and maresin 1 and contributed to tissue regeneration. Ω-3+ LEs protected against hypothermia and weight loss and enhanced survival in murine polymicrobial sepsis. We highlighted a role of Ω-3+ LEs in regulating key mechanisms within the resolution terrain during murine sepsis. This might form the basis for a rational design of sepsis specific clinical nutrition.

Sepsis is associated with a systemic inflammatory reaction, which can result in a life-endangering organ dysfunction. Pro-inflammatory responses during sepsis are characterized by increased activation of leukocytes and platelets, formation of platelet-neutrophil aggregates, and cytokine production. Sequestration of platelet-neutrophil aggregates in the microvasculature contributes to tissue damage during sepsis. At present no effective therapeutic strategy to ameliorate these events is available. In this preclinical pilot study, a novel anti-inflammatory approach was evaluated, which targets nucleoside triphosphate hydrolase activity toward activated platelets via a recombinant fusion protein combining a single-chain antibody against activated glycoprotein IIb/IIIa and the extracellular domain of CD39 (targ-CD39).Experimental animal study and cell culture study.University-based experimental laboratory.Human dermal microvascular endothelial cells 1, human platelets and neutrophils, and C57BL/6NCrl mice.Platelet-leukocyte-endothelium interactions were evaluated under inflammatory conditions in vitro and in a murine lipopolysaccharide-induced sepsis model in vivo. The outcome of polymicrobial sepsis was evaluated in a murine cecal ligation and puncture model. To evaluate the anti-inflammatory potential of activated platelet targeted nucleoside triphosphate hydrolase activity, we employed a potato apyrase in vitro and in vivo, as well as targ-CD39 and as a control, nontarg-CD39 in vivo.Under conditions of sepsis, agents with nucleoside triphosphate hydrolase activity decreased platelet-leukocyte-endothelium interaction, transcription of pro-inflammatory cytokines, microvascular platelet-neutrophil aggregate sequestration, activation marker expression on platelets and neutrophils contained in these aggregates, leukocyte extravasation, and organ damage. Targ-CD39 had the strongest effect on these variables and retained hemostasis in contrast to nontarg-CD39 and potato apyrase. Most importantly, targ-CD39 improved survival in the cecal ligation and puncture model to a stronger extent then nontarg-CD39 and potato apyrase.Targeting nucleoside triphosphate hydrolase activity (CD39) toward activated platelets is a promising new treatment concept to decrease systemic inflammation and mortality of sepsis. This innovative therapeutic approach warrants further development toward clinical application.

The enzyme Δ24-dehydrocholesterol reductase (DHCR24) catalyzes the reduction of the Δ24-double bond in the side chain of cholesterol precursors. Recent biochemical investigations fuel the hope that inhibition of DHCR24, resulting in an accumulation of desmosterol, can open new therapeutic options for treating hepatitis C virus infections, certain forms of cancer and atherosclerosis. In turn, there is a high need for selective, potent and non-toxic inhibitors of DHCR24. Previous reports as well as our re-evaluation showed that established DHCR24 inhibitors are not suitable for this purpose. Based on the lathosterol-derived amide MGI-21 (IC50 823 nM for inhibition of overall cholesterol biosynthesis in HL-60 cells) we performed a systematic variation of the side chain functionality and identified the steroidal 3,22-diols 29 and 30, as well as several esters thereof, as extremely potent (IC50

The resolution of inflammation is an active process that is coordinated by endogenous mediators. Previous studies have demonstrated the immunomodulatory properties of the axonal guidance proteins in the initial phase of acute inflammation. We hypothesized that the neuronal guidance protein neogenin (Neo1) modulates mechanisms of inflammation resolution. In murine peritonitis, Neo1 deficiency (Neo1-/-) resulted in higher efficacies in reducing neutrophil migration into injury sites, increasing neutrophil apoptosis, actuating PMN phagocytosis, and increasing the endogenous biosynthesis of specialized proresolving mediators, such as lipoxin A4, maresin-1, and protectin DX. Neo1 expression was limited to Neo1-expressing Ly6Chi monocytes, and Neo1 deficiency induced monocyte polarization toward an antiinflammatory and proresolving phenotype. Signaling network analysis revealed that Neo1-/- monocytes mediate their immunomodulatory effects specifically by activating the PI3K/AKT pathway and suppressing the TGF-β pathway. In a cohort of 59 critically ill, intensive care unit (ICU) pediatric patients, we found a strong correlation between Neo1 blood plasma levels and abdominal compartment syndrome, Pediatric Risk of Mortality III (PRISM-III) score, and ICU length of stay and mortality. Together, these findings identify a crucial role for Neo1 in regulating tissue regeneration and resolution of inflammation, and determined Neo1 to be a predictor of morbidity and mortality in critically ill children affected by clinical inflammation.

Pancreatic enzyme preparations are a life-saving substitution for a pivotal physiological function of the entire organism that is impaired in chronic pancreatitis, cystic fibrosis and other diseases with exocrine pancreatic insufficiency. Pancreatic enzyme preparations, generically called pancreatin, are not alike. Rather, they present a broad variety of pancreatin composition.The properties of a set of commercially available pancreatin preparations were investigated in light of the physiological tasks such enzymes must fulfill during the normal digestive process.Measurements of size, surface, acid resistance, release of enzymes, pharmacokinetics and batch consistency were undertaken.Although all pancreatin preparations contain the declared lipase units and are acid-stable, a wide variation was observed in the particle size (pyloric passage), specific surface area and release kinetics of lipase activity at pH 6 (duodenum).At present, available pancreatin preparations vary widely with respect to investigated parameters, which may have consequences for facilitating optimal digestion.

Hepatic ischemia/reperfusion (I/R) is a major adverse reaction to liver transplantation, hemorrhagic shock, or resection. Recently, the anti-inflammatory properties of the axonal guidance cue netrin-1 were reported. Here, we demonstrate that netrin-1 also impacts the resolution of inflammation and promotes hepatic repair and regeneration during liver I/R injury. In initial studies, we investigated the induction of netrin-1 and its receptors in murine liver tissues after I/R injury. Hepatic I/R injury was performed in mice with a partial genetic netrin-1 deficiency (Ntn1(+/-)) or wild-type C57BL/6 treated with exogenous netrin-1 to examine the endogenous and therapeutically administered impact of netrin-1. These investigations were corroborated by studies determining the characteristics of intravascular leukocyte flow, clearance of apoptotic neutrophils (polymorphonuclear cells [PMNs]), production of specialized proresolving lipid mediators (SPMs), generation of specific growth factors contributing to the resolution of inflammation, and liver repair. Hepatic I/R was associated with a significant reduction of netrin-1 transcript and protein in murine liver tissue. Subsequent studies in netrin-1-deficient mice revealed lower efficacies in reducing PMN infiltration, proinflammatory cytokine levels, and hepatic-specific injury enzymes. Conversely, mice treated with exogenous netrin-1 exhibited increased liver protection and repair, reducing neutrophil influx into the injury site, decreasing proinflammatory mediators, increasing efferocytosis of apoptotic PMNs, and stimulating local endogenous biosynthesis of SPMs and the generation of specific growth factors. Finally, genetic studies implicated the A2B adenosine receptor in netrin-1-mediated protection during hepatic I/R injury. Conclusion: The present study indicates a previously unrecognized role for netrin-1 in liver protection and its contribution to tissue homeostasis and regeneration. (Hepatology 2016;63:1689-1705)

OBJECTIVE Previous studies of sex hormone concentrations in depression yielded inconsistent results. However, the activation of the hypothalamic-pituitary-adrenal system seen in depression may negatively affect gonadal function at every level of regulation. The objective of this study was to explore whether major depressive episodes are indeed associated with an alteration of gonadal function. METHODS Testosterone, pulsatile LH secretion, FSH, and cortisol were assessed using frequent sampling during a 24-hour period in 15 male inpatients with major depression of moderate to high severity and in 22 healthy comparison subjects (age range 22-85 years). RESULTS An analysis of covariance model showed that after adjustment for age only, daytime testosterone (p < .01), nighttime testosterone (p < .05), and 24-hour mean testosterone secretion (p < .01) were significantly lower in the depressed male inpatients. There was also a trend for a decreased LH pulse frequency in the depressed patients (p < .08). CONCLUSIONS Gonadal function may be disturbed in men with a depressive episode of moderate to high severity.

There is compelling evidence for feedback disturbances in the hypothalamus-pituitary-adrenal system associated with human aging as assessed by challenge tests. However, reports about age-related changes in human basal activity are ambiguous and to date little is known about changes in the pulsatile features of the HPA system. To investigate these changes we studied twenty-two healthy male and eleven healthy female subjects ranging from 23 to 85 and 24 to 81 years respectively. 24-hour blood sampling with 30 minute sampling intervals was performed. From 18.00 to 24.00 hours blood was sampled every 10 minutes for analysis of pulsatile features of HPA activity. Statistical analysis revealed that age in particular had major effects upon basal HPA-system activity: there was a significant age-associated increase in minimal (p < 0.0001) and mean (p < 0.02) cortisol plasma concentrations, but no alteration in pulsatile features. We found no age-cortisol correlation during daytime, but were able to demonstrate a strong impact of age upon cortisol plasma levels from 20.00 to 1.30 hours. The diurnal amplitude of cortisol (p < 0.005) and ACTH (p < 0.006), relative to the 24-hour mean of the hormones, showed an age-associated decline. Additionally, the evening cortisol quiescent period (p < 0.01) was shortened in the elderly, suggesting increasingly impaired circadian function in aging. Our results suggest an increased basal activity and a flattened diurnal amplitude of the HPA system in the elderly.

It has been shown that a single pulse-dosing (PD) dose of clomipramine improves depressive symptoms. However, so far PD and conventional (CONV) application of antidepressants have never been directly compared for an extended period. We performed a double-blind study of PD and CONV application of doxepin (DOX) in depressed patients. After a 1-week placebo treatment, nine parents in the PD group received 250 mg of DOX every 6 days and placebo on the other days until day 39. Ten patients in the CONV group received increasing dosages of DOX until day 7 and 250 mg DOX on the other days for 39 days. Three dexamethasone (DEX)-suppression/corticotropin-releasing hormone (CRH)-stimulation tests were completed: (1)during the initial placebo period; (2)on day 9; and (3)on day 21. In the PD group, scores on the Hamilton Rating Scale for Depression (HAM-D) differed from baseline only after day 36 (17.1 +/- 7.0 vs. 22.7 +/- 2.8, p < 0.03). In the CONV group, however, HAM-D scores improved significantly after 2 days (22.8 +/- 7.2 vs. 26.5 +/- 5.7, p < 0.02) and continued to improve until day 39 (7.3 +/- 5.8). From day 25 to 39, there were significant differences between the HAM-D scores of the two groups. In the PD group, the decline of cortisol after DEX pretreatment was nonsignificant (NS) at both follow-up test occasions (35.9 +/- 40.7 vs. 24.0 +/- 20.7 vs. 23.6 +/- 26.6 micrograms/mL). In the CONV group, a significant decrease was observed at the second test (61.8 +/- 61.9 vs. 10.7 +/- 4.2 vs. 19.8 +/- 19 micrograms/mL, p < 0.05, respectively, NS). The area-under-the-curve cortisol response after CRH was attenuated in the PD group (5,667 +/- 2,910 vs. 1,883 +/- 2,178 vs. 2,239 +/- 2,583 [arbitrary unit], p < 0.01, respectively, p < 0.01) and in the CONV group (5,710 +/- 4,734 vs. 1,267 +/- 2,053 vs. 445 +/- 1,016 [arbitrary unit], NS, respectively, p < 0.02. We conclude that CONV application of DOX is clinically superior compared with PD and that both modes of application have attenuating effects on hypothalamus-pituitary-adrenal system activity.

CYP2D6 is part of the cytochrome P450 system, which catalyzes biotransformation of endogenous substrates and xenobiotics. Approximately 10% of the Caucasian population has two null alleles, resulting in a poor metabolizer (PM) status. Mostly, allele four (CYP2D6*4) is responsible for the PM status, which is suspected to be associated with an accelerated fibrosis progression (FP). The aim of the present study was to analyze the role of the CYP2D6*4 genotype for FP after liver transplantation (LT). Genotypes were determined in liver biopsies (donor) and peripheral blood (recipient) by fluorescence resonance energy transfer. Data were correlated with clinical variables and risk factors for fibrosis. We analyzed 517 LTs performed between 1997 and 2009. Overall donor and recipient allele frequencies were comparable (18.0%, 20.5%; P = 0.43). The donor genotype did not correlate with FP. In contrast, recipients carrying CYP2D6*4, showed a significant higher risk for an accelerated FP (P = 0.011) in HCV positive (P = 0.038) and HCV negative patients (P = 0.033). Results were confirmed by multivariate analysis (Hazard ratio 1.65; P = 0.001). The CYP2D6*4-associated PM status of the donor liver seems to have no influence on FP after LT. Recipients, carrying the allele, have an elevated risk for an accelerated FP.

There is an age-associated decline in the activity of the GH-IGFs system. However, so far, it has not been studied, whether this decline in somatotrophic activity might be preventable by intensive exercising. We studied 11 elderly male (50-78 years) marathon runners and 10 age-matched male (52-73 years) sedentary controls to evaluate plasma concentrations of GH, total and free IGF-I and IGF-II and of IGF-binding protein-1 (IGFBP-1), IGFBP-2, IGFBP-3 and insulin. When comparing the two groups (runners vs controls) no differences were found in GH (1.0 +/- 1.2 vs 1.3 +/- 1.3 microg/l [mean +/- SD]), IGF-1 (115 +/- 23 vs 113 +/- 21 microg/l), IGF-II (961 +/- 192 vs 864 +/- 125 microg/l), free IGF-1 (227 +/- 80 vs 318 +/- 146 ng/l), free IGF-II (563 +/- 249 vs 492 +/- 108 ng/l), IGFBP-3 (2403 +/- 515 vs 2307 +/- 326 microg/l) or insulin (26 +/- 13 vs 27 +/- 18 mU/l). However, IGFBP-1 (4.44 +/- 2.61 vs 2.28 +/- 0.93 microg/l) and IGFBP-2 (493 +/- 143 vs 340 +/- 186 microg/l) were found to be significantly increased in marathon runners. In conclusion, our findings do not support the hypothesis that the age-associated decline in GH, IGF-1 and IGFBP-3 may be preventable by intensive endurance training. However, marathon running affects the regulation of the GH-IGFs system activity at the level of IGFBP-1 and -BP-2.

Background: The dexamethasone suppression test (DST) is a widely used endocrine test in psychiatry, but was reported to not allow reliable inferences with regard to the basal activity of the Inpothalamo-pituitary-adrenocorti-cal (HPA) system. We compared the association of the standard DST and the combined dexamethasone/cortico-tropin-releasing hormone (DEX/CRH) challenge with parameters of diurnal Cortisol profiles. Methods: We performed a DEX/CRH challenge and 24-hour Cortisol profiles in 25 depressed patients (mean age: 47.4 ± 16.0 years) and 33 age-matched healthy controls (mean age: 51.4 ± 19.3 years). Results: A path analysis showed Cortisol area under the curve (AUC) after CRH (= DEX/CRH status) to be dependent upon minimal 24-hour Cortisol and evening frequency of pulsatile Cortisol release. In contrast, post-dexamethasone Cortisol (= DST status) was related to 24-hour mean Cortisol. Simple linear regressions supported an association of Cortisol AUC with several parameters of the diurnal Cortisol profiles, which was not true for the standard DST. Conclusions: We conclude that the combined DEX/CRH challenge test is more closely associated with the activity of the HPA system than the standard DST in healthy and depressed subjects.

There is some evidence that the somatotrophic system in depression, as assessed by basal growth hormone (GH) concentrations and by GH releasing hormone (GHRH) challenge, might be dysfunctional. However, the rather limited data have been inconclusive so far and plasma concentrations of both insulin-like growth factor-1 (IGF-I) and binding proteins (IGFBP 1 to IGFBP-6) have not been measured simultaneously in depressed patients. We studied 24 severely depressed patients and 33 healthy controls and estimated 24-hour mean plasma cortisol, six-hour evening mean plasma growth hormone (GH), morning plasma IGF-I, IGFBP 2 and 3 and GH-binding protein (GH-BP). Twenty-four-hour mean cortisol (306 +/- 69 vs. 196 +/- 30 nmol/l, p < .001) and IGF-I (157 +/- 40 vs. 120 +/- 33 micrograms/l, p < .01) plasma concentrations were found to be significantly increased in depressed patients, while there was no difference in GH or binding proteins between both groups. MANOVA analysis revealed age and diagnosis to have main effects upon plasma IGF-I. Especially young age and a diagnosis of major depression are associated with higher plasma IGF-I. After treatment only patients in remission had attenuated IGF-I plasma concentrations. We conclude that plasma IGF-I is increased in acutely depressed patients similar to other states of hypercortisolemia.

There is only sparse and ambiguous information about circadian and pulsatile secretion features of the hypothalamus-pituitary-adrenocortical system in depression. We studied 15 severely depressed (Hamilton Depression Scale 30.4 +/- 6.7) male patients (age 22-72 yr; mean, 47.7 +/- 14.8) and 22 age-matched male controls (age 23-85 yr; mean, 53.1 +/- 18.2). Twenty-four-hour blood sampling from 0800-0800 h with 30-min sampling intervals was performed; from 1800-2400 h, blood was drawn every 10 min. Multivariate analysis of covariance, with the covariate being age, revealed mean 24-h cortisol (315.9 +/- 58.5 vs. 188.2 +/- 27.3 nmol/L) and mean ACTH (7.82 +/- 1.94 vs. 5.79 +/- 1.28 pmol/L) to be significantly increased in depressed patients. The frequency of cortisol (2.6 +/- 0.7 vs. 1.3 +/- 1.0 pulses/6 h) and ACTH (2.6 +/- 1.6 vs. 1.6 +/- 1.4 pulses/6 h) pulses during the evening were higher in patients compared to controls. The flattened circadian cortisol variation and reduced time of quiescence of cortisol secretory activity (140 +/- 116 vs. 305 +/- 184 min) in patients suggest disturbances of circadian functions. We conclude that increased hypothalamus-pituitary-adrenocortical activity in depression is related to a greater frequency of episodic hormone release, and we hypothesize that the observed circadian changes might be partly due to altered mineralocorticoid and glucocorticoid receptor capacity and function.

The pharmacokinetics of amitriptyline (AMI) have been extensively studied, and a large interindividual variability between oral dose and concentration of the drug in plasma has been documented. The aim of this study was twofold: first, to compare AMI kinetics in depressed patients with those of healthy controls and, second, to describe the relationship between AMI levels in plasma and hypothalamic-pituitary-adrenal (HPA) system changes during depression. Thirty-eight patients with a DSM-III-R diagnosis of major depression and 13 healthy control persons received 75 mg of AMI daily for 6 weeks. Levels of AMI and nortriptyline in plasma were determined, and neuroendocrine testing with the combined dexamethasone-suppression/CRH-stimulation test (DST) was done before AMI administration and after weeks 1, 3, and 6 of medication. AMI levels in plasma were significantly higher in the patient group compared with controls during the entire treatment period, whereas nortriptyline levels did not differ between the two groups. Drug levels correlated significantly with age, but gender had no effect on the concentration of the drug in plasma. Twenty-two patients remitted after treatment. There was no difference in drug levels between responders and nonresponders. Fifteen patients were DST nonsuppressors before treatment; 23 patients and all controls suppressed cortisol after dexamethasone. DST suppressors had significantly higher AMI levels in plasma at weeks 3, 5, and 6 compared with DST nonsuppressors. In comparison to patients with high AMI levels in plasma, those with low drug concentration had higher postdexamethasone cortisol and adrenocorticotropic hormone levels and an increased hormone release after additional CRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Major depression is associated with hypercorticoidism, a risk factor for osteoporosis. However, it is unknown whether depressive disorders are associated with alterations in bone mineral density. The authors measured the density of trabecular bone from the first to the third lumbar vertebra by quantitative computerized tomography in 80 depressed inpatients older than 40 years and in 57 healthy comparison subjects. An analysis of covariance model with age as a covariate showed a significant effect of diagnosis on the dependent variable spinal bone mineral density: depressed patients had lower values. Other factors could not explain the finding. The authors conclude that major depression is a significant risk factor for osteoporosis.