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Inflamm. Res.Inflammation Research Inflammatory responses improve with milk ribonuclease-enrichedlactoferrin supplementation in postmenopausal women Satish Bharadwaj • Tezus A. G. Naidu •Guru V. Betageri • Nemani V. Prasadarao •A. Satyanarayan Naidu Received: 16 October 2009 / Revised: 14 January 2010 / Accepted: 27 April 2010Ó Springer Basel AG 2010 CRP were modestly reduced (-50%) relative to their pla- Objective and design A 6-month, randomized clinical cebo levels, although RANKL elevated initially.
study was conducted to evaluate the effect of a ribonu- R-ELF supplementation showed beneficial clease-enriched lactoferrin (R-ELF) supplement on the effects towards improvement of inflammatory status in circulating cytokine levels and bone health of postmeno- Thirty-eight healthy postmenopausal women, Lactoferrin Osteoporosis Postmenopausal aged 45–60 years, were randomized into placebo and Ribonuclease Inflammation The R-ELF group was supplemented with R-ELF (2 9 125 mg/day) and calcium (100% RDA), while the placebo group received only the calcium supplement.
Methods Serum levels of receptor activator for NF-jB Normal physiological conditions ensure a balance between ligand (RANKL), C-reactive protein (CRP) and various bone formation and bone resorption to maintain skeletal pro- and anti-inflammatory cytokines were determined by homeostasis. An imbalance develops during osteoporosis and age-related osteopenia in postmenopausal women Pro-inflammatory cytokines IL-6 and TNF-a Excess production of osteoclasts and inadequate generation decreased significantly (-44 and -10%, respectively) of osteoblasts could lead to such homeostatic imbalance.
while anti-inflammatory IL-10 increased (140%) due to Current treatment options for postmenopausal osteoporosis R-ELF supplementation at the end of study. RANKL and include hormone replacement therapy (HRT), selectiveestrogen receptor modulators (SERM) and bisphospho-nates. HRT is effective in reducing osteoclast activity, Responsible Editor: Ian Ahnfelt-Rønne.
however, with certain potential detrimental effects includ-ing increased risk of breast cancer and thrombo-embolism S. Bharadwaj T. A. G. Naidu A. S. Naidu (&) as reported in the Women's Health Initiative [] and the N-terminus Research Laboratory, 981 Corporate Center Dr., Million Women Study []. SERMs such as raloxifene are # 110, Pomona, CA 91768, USAe-mail: [email protected] known to disrupt lymphocyte development, immune cellfunction, and cytokine secretion A risk for develop- ment of esophageal ulcers has also been reported with the Department of Pharmaceutical Sciences, College of Pharmacy, use of bisphosphonates Emerging anabolic strategies Western University of Health Sciences, 309 E Second Street,Pomona, CA 91766, USA include strontium renelate, isosorbide mononitrate andteriparatide (synthetic parathyroid hormone) that show a positive effect on bone formation as well as a reduction in Division of Infectious Diseases, Childrens Hospital Los Angeles bone resorption activity However, these new treat- and Keck School of Medicine, University of Southern California,4650 Sunset Blvd., Los Angeles, CA 90027, USA ments require further safety evaluation ]. Driven by the S. Bharadwaj et al.
imperative to explore and develop strategies for enhancing Materials and methods bone formation by natural products and simultaneouslyprevent bone loss without side effects, we recently reported the development of a milk-ribonuclease-enriched lactofer-rin (R-ELF) supplement and its effect on bone turnover in Selection of subjects was based on the response to a postmenopausal women ]. The data revealed that questionnaire on general bone health status, previous R-ELF reduced bone resorption and increased bone for- injury/disease, current or previous treatment, and con- mation significantly. Nonetheless, the effect of R-ELF on sumption of calcium-rich foods. General health was general inflammation was not evaluated.
determined by routine standard medical assessment of The immune and the skeletal systems share several physical and mental health. Details of the inclusion and regulatory factors, such as cytokines, transcription factors, exclusion criterion for the study have been described pre- and receptors. The immune cells and osteoclasts are viously ]. After evaluation, 38 healthy, ambulatory derived from the same hematopoietic precursor cells, postmenopausal women, 45–60 years old, with no menses which originate in bone marrow and interact with bone for at least 12 months were registered for the study. Three cells , ]. Inflammation is the primary defense mech- subjects were excluded, one with a history of treatment for anism of the body, characterized by a ‘high alert' state of bone health and two women had hypothyroidism. The the immune system, triggered by the release of several pro- study was approved by the Institutional Review Board of inflammatory cytokines. It also affects the processes of the Western University of Health Sciences, Pomona, CA.
bone resorption and formation, due to the presence of Prospective participants were advised of the nature of the pathways that are common to both bone cell maturation study and provided written informed consent before and inflammation. Pro-inflammatory cytokine levels are known to rise during aging and stress ]. Elevated levelsof tumor necrosis factor (TNF)-a, interleukin (IL)-6 and IL-1b receptors suggest subtle changes in the immunesystem [Thus, the body is in a state of mild, continuous R-ELF is a ribonuclease (angiogenin)-enriched LF either inflammation, characterized by a rise in the levels of acute co-isolated from bovine milk (50:50 ratio wt/wt) or both phase proteins such as C-reactive protein (CRP), a well- proteins (LF and ribonuclease) admixed to obtain required known bio-marker for inflammation. In healthy, elderly ratios, as previously described [Thirty-five women individuals higher serum CRP levels were associated with included in the study were randomly assigned to one of the high bone turnover rate, resulting in low bone mineral two groups: placebo group or R-ELF group. Fifteen sub- jects assigned to the placebo group were supplemented Lactoferrin (LF) is a multifunctional milk protein with with 100% RDA (Recommended Daily Allowance) of anti-inflammatory and immune modulatory properties calcium, in a tablet form, whereas 20 subjects of the R-ELF [LF stimulates lymphocytes and natural killer group were given two R-ELF capsules of 125 mg each, cells, and is involved in multiple pathways of immune along with 100% RDA of calcium administered orally from response. Enhanced secretion of the anti-inflammatory Day 1 to Day 180. Venous blood samples were collected, cytokines IL-4 and IL-10, along with altered expression by standard venipuncture technique, from each subject on of pro-inflammatory cytokines (IFN-c, IL-1b, IL-6 and Day 0 (baseline before starting the supplement), Day TNF-a) have been observed with LF intake []. Oral 30, Day 90 and Day 180 of the study. Further details of administration of LF in animal models could upregulate design and flow of the study have been described previ- anti-inflammatory IL-4 and IL-10 [and inhibit TNF-a along with a rise in IL-10 secretion in animal arthritismodels Inflammatory cytokines and markers and anti-inflammatory properties of LF and the estab-lished links between inflammatory cytokines with bone The levels of inflammatory cytokines—IFN-c, IL-6, IL- remodeling processes, we set out to investigate the 12?p40, TNF-a, IL-1b, IL-10 and TGF-b were determined changes in inflammatory status of subjects supplemented by enzyme-linked immunosorbent assay (ELISA) using with R-ELF. The present study reports the effect of respective monoclonal antibodies purchased from Invitro- R-ELF supplementation on the levels of inflammatory gen (Camarillo, CA). High purity CRP and anti-CRP rabbit cytokines and CRP, as indicators of inflammation, and polyclonal antibody were obtained from Calbiochem/EMD receptor activator for nuclear factor-jB ligand (RANKL) (San Diego, CA, USA) and anti-RANKL rabbit polyclonal as a marker of osteoclast activity in postmenopausal antibody was from Abcam Inc. (Cambridge, MA, USA).
CRP assay had a sensitivity of 8 pg/mL and detection limit Inflammatory responses in postmenopausal women of 1 ng/mL. All buffers and other reagents were obtained health characteristics of the placebo and R-ELF groups, from Invitrogen.
reflecting generally good bone health status. As detailed inour previous study, the bone health status of the two groups Statistical analysis were also comparable, as evaluated by measuring the levelsof several bone turnover markers ]. Three participants Cytokine data from each day was analyzed for measures of from the control group dropped out of the study before Day central tendency, deviation and distribution of data. Data 15 and one from the R-ELF group was dropped due to non- were considered outliers if they were [1.5 times the inter- compliance. There was [95% compliance to the supple- quartile range (IQR) above the third quartile or below the ment regimen among the subjects. The body weight and first quartile. Outliers were discarded from datasets for blood pressure of all the subjects essentially maintained statistical tests of significance. In view of the small size of within ±3% of their baseline values. No adverse events placebo and R-ELF groups, median and standard error of were reported during the 6-month study or 3-month post- the mean were used as the preferred measures of central study follow up.
tendency for this modest set of data. The Kolmogorov– The mean levels and range of inflammatory cytokines in Smirnov test (KS test) was used as a test for normal dis- the serum of study subjects were similar to those reported tribution of data within each set Student's unpaired for generally healthy individuals []. The levels of two-sample t test was used for comparison of the mean TGF-b in postmenopausal women are known to vary observed change in markers for placebo and R-ELF data widely, ranging from 9,800 ] to 46,500 ]. In the sets to establish the effect of R-ELF supplementation.
analysis of cytokine levels, the median ± SEM values are OriginPro Ver. 8 (OriginLab, MA, USA) software was used to estimate changes with R-ELF supplementation, as used for data analysis.
the groups were small. Unusual variations in the data ofsome subjects resulted in high standard errors.
Pro-inflammatory cytokine levels improved towardsa better bone health Baseline characteristics, compliance and adverse events Variation in the serum levels of IFN-c, IL-1b, TNF-a, IL-6 The baseline characteristics of placebo and R-ELF groups and IL-12?p40 for placebo and R-ELF groups during the are shown in Table . There is a close match between the course of the study are shown in Fig. The median IFN-cranged from 14.4 ± 1.7 to 18.3 ± 2.9 pg/mL for the pla-cebo group while the values increased from 28.6 ± 13.7 to Table 1 Baseline characteristics of the study population 32.4 ± 12.8 pg/mL for the R-ELF group and the difference between them was statistically significant (P = 0.0007for median IFN-c, placebo vs. R-ELF). IFN-c levels for Number of participants (n) the R-ELF group decreased (-6.6%) within 30 days but increased considerably (?42%) for the placebo group. By the end of study, although IFN-c levels for the R-ELF group were increased (?13.5%), the increment was smaller compared to the placebo group (?27%) (Fig. a). Median IL-1b levels ranged from 10.2 ± 3.8 to 15.0 ± 10.2 pg/mL for the placebo and from 22.6 ± 10.9 to 23.3 ± 19.9 Blood pressure (mm Hg) pg/mL for the R-ELF group (P = 0.0131 for median Mean Systolic ± SD IL-1b, placebo vs. R-ELF). A large decrease in IL-1b Mean Diastolic ± SD levels was observed initially but the trend reversed by Day Cytokines (pg/mL)a 90 and turned out significantly positive (?46.5%) for the placebo group, while it remained close to the baseline (?2.8%) for the R-ELF group (Fig. TNF-a levels ranged from 239.3 ± 15.8 on Day 0 to 238.3 ± 69.5 pg/mL for placebo, while a decrease from 279.7 ± 31.0 to 251.4 ± 43.2 pg/mL (P = 0.0341 for median TNF-a, placebo vs. R-ELF) was observed with R-ELF supple- mentation. A large initial decline in TNF-a levels (-27.4% for R-ELF vs. -15.5% for placebo) was sustained until
S. Bharadwaj et al.
Fig. 1 Effect of R-ELFsupplementation on pro-inflammatory cytokines. Medianchange in the serum levels ofpro-inflammatory cytokines.
IFN-c (a), IL-1b (b), TNF-a (c),IL-6 (d) and IL-12 (e)—insubjects from placebo (openbars) and R-ELF (filled bars)supplemented groups
Day 180 for the R-ELF group (-10.1%), but not with the
TGF-b were determined for both groups and are shown in
placebo (-0.4%) (Fig. Furthermore, median IL-6
Fig. Levels of IL-10 for the placebo group were close to
levels slightly decreased for the placebo group from 2.1 ±
the baseline median value of 17.4 ± 2.7, until Day 180
1.5 on Day 0 to 2.0 ± 2.1 pg/mL by the end of study,
(16.3 ± 3.6 pg/mL), while the levels increased from
while IL-6 decreased from 5.5 ± 3.9 to 3.0 ± 4.1 pg/mL
7.7 ± 9.2 to 18.8 ± 4.2 pg/mL for the R-ELF group. A
for the R-ELF group. As shown in Fig. by the end of
major positive change was observed in IL-10 with R-ELF
study, the decrease in IL-6 was significant (P = 0.0338
supplementation (?150%) compared to placebo (-6.2%)
for median IL-6, placebo vs. R-ELF) for the R-ELF
(P = 0.0235 for change in median IL-10, placebo vs.
group (-44.1%) compared to the placebo (-4.3%). Levels
R-ELF) by the end of study (Fig. Median TGF-b levels
of IL-12?p40 (also referred to as IL-12) were similarfor both groups; placebo ranging from 68.5 ± 1.6 to90.2 ± 1.6 pg/mL and R-ELF group from 73.1 ± 4.0 to aslightly higher 111.1 ± 5.6 pg/mL. R-ELF supplementa-tion significantly raised IL-12 levels by 51.9% by Day 180compared to placebo, with an increase of 31.8% from theirrespective baseline levels (P 0.0005 for mean IL-12,placebo vs. R-ELF) (Fig. These results demonstratethat R-ELF supplementation induced down-regulation ofpro-inflammatory TNF-a and IL-6, and a moderate eleva-tion of IL-1b and IFN-c levels within 6 months.
Effect of R-ELF supplementation on anti-inflammatorycytokines
Enhanced production of anti-inflammatory cytokines, such
Fig. 2 Variations in serum levels of anti-inflammatory cytokineswith R-ELF supplementation. Median change in the serum levels of
as IL-10 and TGF-b, would complement the beneficial
anti-inflammatory cytokines. IL-10 (a) and TGF-b (b) in subjects
effects of R-ELF as these cytokines promote formation of
from placebo (open bars) and R-ELF (filled bars) supplemented
new bone matrix. Changes in the serum levels of IL-10 and
Inflammatory responses in postmenopausal women
slightly decreased for both the groups, 786.3 ± 73.1 to761.5 ± 85.1 pg/mL for the placebo, compared to 739.4 ±58.9 to 727.6 ± 51.9 pg/mL for the R-ELF (P = 0.0072for median TGF-b, placebo vs. R-ELF). However, thedecrease was smaller for the R-ELF group (-2%) than theplacebo (-3%) (Fig. b). These results indicate a relativelypositive change in anti-inflammatory cytokines withR-ELF supplementation, compared to calcium supple-mentation alone.
The correlation between pro- and anti-inflammatory
cytokines was analyzed and the results are presented inTable . The correlation matrix for placebo and R-ELFgroups were generally maintained from Day 0 to Day 180and improved with R-ELF supplementation. Most cytokinecorrelations also remained statistically significant at 95%
Fig. 3 Changes in serum levels of RANKL and CRP with R-ELFsupplementation. Median change in the serum levels of RANKL, a
confidence interval. The correlation of IL-6 versus IL-10
marker of osteoclast formation (a) and CRP, an inflammatory marker
improved with R-ELF supplementation. In general, TNF-a,
(b) in subjects from placebo (open bars) and R-ELF (filled bars)
IL-12?p40 and TGF-b did not show any correlation, nei-
ther with the progress of study nor with supplementation.
IL-10 was highly correlated with IFN-c and IL-6 at the
Reduced levels of CRP and RANKL with R-ELF
beginning of the study for both groups, which improved by
the end of the study. Interestingly, IFN-c was not correlatedwith IL-10 at baseline but demonstrated a high degree of
RANKL is a well established marker for the onset of
correlation after R-ELF supplementation. These positive
osteoporosis in postmenopausal women. RANKL, a
correlations suggested a moderate elevation of anti-
mediator of osteoclast formation, indicated a sharp rise by
inflammatory cytokines that could reduce pro-inflamma-
Day 30 followed by a steady decline for both placebo and
tory response. Inverse correlation is a favorable outcome of
R-ELF groups (Fig. However, in response to R-ELF
reduction in pro-inflammatory and elevation of anti-
supplementation there was a larger and rapid decline by
inflammatory cytokines, in which could diminish inflam-
-34.8% (60.6% on Day 30 to 25.8% on Day 90) compared
mation. An improved negative correlation was observed
to an 11.6% increase (35.0% on Day 30 to 46.6% on Day
between IL-6 and TGF-b with R-ELF supplementation,
90) with calcium supplementation alone. By the end of the
although not statistically significant.
study, RANKL levels were further decreased to 8.2% forR-ELF group and remained at 22.5% for placebo(P = 0.0202 for median change in RANKL, placebo vs.
R-ELF). Similarly, CRP levels showed a sharp decrease for
Table 2 Correlation between pro- and anti-inflammatory cytokines
both the groups by Day 30, but the trend reversed and CRP
increased to baseline levels by the end of the study
(Fig. b). A relatively large reduction in CRP levels wasobserved for the R-ELF group (-55.9% by Day 30), which
sustained for a long time (-49.8% by Day 90) (P = 0.0286
0.97 ( 0.0005) nc
for mean change in CRP, placebo vs. R-ELF). On the other
0.97 ( 0.0005) nc
hand, the placebo group regained CRP levels rapidly,
-40.6 to -23.1% of its baseline CRP level in 60 days.
0.98 ( 0.0005) nc
These results suggest an improvement in the inflammatory
status and reduced bone resorption in postmenopausal
women with R-ELF supplementation.
-0.41b (0.271) 0.92 ( 0.05)
nc No correlation observed
In our previous study, R-ELF supplementation reduced
a 90% Confidence interval
bone resorption and significantly elevated bone formation,
b Not statistically significant
measured as changes in the levels of several bone turnover
S. Bharadwaj et al.
markers [Bone metabolism is known to be influenced
placebo, indicating a beneficial effect. IL-1b, a potent
by inflammatory cytokines. Development and activation of
stimulator of osteoclastogenesis however, showed an
osteoclasts and subsequent bone resorption is regulated by
initial decrease followed by an increase by Day 90, then
the RANKL pathway []. Anti-resorptive therapies,
settled close to baseline by the end of study despite R-ELF
such as denosumab, targeted towards RANKL, are cur-
rently being developed ]. Several cytokines influence
increased by [40% by Day 180 in the placebo group,
the expression of RANKL, which in turn, affects bone
whereas R-ELF supplementation prevented such an
remodeling ]. Estrogen deficiency in postmenopausal
increase, indicating that long term supplementation of
women causes elevation of pro-inflammatory cytokines
R-ELF may be necessary to observe positive effects. In
such as TNF-a and IL-6, which increases RANKL levels,
contrast, IL-12 inhibits the RANKL-induced maturation
leading to increased osteoclast maturation, activity and
and activation of osteoclasts on its own or in synergy with
subsequent risk for osteoporotic fractures []. Other
IL-18 ]. R-ELF increased the serum levels of IL-12 by
treatments, such as HRT and raloxifene, resulted in either
more than 50% in 6 months, which may be responsible for
increased or unchanged levels of IL-6, although TNF-a
the observed decrease in RANKL levels after Day 30.
levels showed a considerable decline. –In the
Further, IL-12 has been shown to inhibit TNF-a induced
present study, we have observed a significant reduction in
osteoclastogenesis via T cell dependent mechanism in mice
TNF-a and IL-6 levels, after six months of R-ELF sup-
]. Therefore, the increased levels of IL-12 with R-ELF
plementation, with substantial favorable changes in TNF-a
supplementation are a favorable outcome in this study.
(-27%) and IL-6 (-44%) occurring within the first
Anti-inflammatory cytokines, TGF-b and IL-10, tend to
90 days. Based on this decrease in TNF-a and IL-6 levels,
suppress osteoclastogenesis [–A reduction in
a change in RANKL was expected in R-ELF treated sub-
TGF-b levels results in inflammation, autoimmunity and
jects. However, median change in RANKL levels varied
increased T cell production of IFN-c, TNF and RANKL. In
significantly from baseline and was reduced for the R-ELF
the present study, TGF-b levels for the placebo group
group relative to placebo by the end of study. It is note-
exhibited slight decrease (-3%) whereas R-ELF supple-
worthy that RANKL levels have not shown a consistent
mentation limited the loss to -2%. The change in TGF-b
behavior with the progress of anti-resorptive therapy.
levels is minimal, however, serum TGF-b levels were
Similar inconsistent variations in RANKL levels have been
significantly lowered by HRT ([50%) while raloxifene
well documented –In serum, RANKL is present in
treatment had no effect [The marked increase in IL-10
free form as well as bound to osteoprotegerin (OPG). The
levels (?150%) observed with the R-ELF group appears
increase in RANKL levels has been interpreted as an
also to be a beneficial outcome. In view of the detrimental
increase in the OPG-bound form, which is a favorable
effects of IL-10 deficiency, IL-10 elevation by R-ELF
outcome, indicating reduced osteoclast activity ]. In
could be responsible for the increased bone formation (as
addition, RANKL levels were undetectable for several
measured by bone specific alkaline phosphatase and oste-
subjects in a raloxifene study due to limitation of the assay
ocalcin levels) observed in our previous clinical study [
[Therefore, our preliminary observations considering
The kinetics of acute phase marker CRP synthesis are
the results of other investigators, suggest that R-ELF
influenced by several cytokines involved in the inflamma-
intervenes with RANK/RANKL pathway, directly or
tory process. The serum concentration of CRP has been
indirectly, to down-regulate osteoclast formation and sub-
shown to correlate with TNF-a and IL-6 with minor effect
sequently reduce bone resorption (as has been observed by
from other cytokines [R-ELF supplement, therefore,
changes in serum NTx and urine DPD markers in our
was expected to bring down inflammation considerably
previous study []).
with reduction of TNF-a and IL-6 levels. In agreement, the
The pro-inflammatory cytokine IFN-c has a dual role in
R-ELF group demonstrated significantly lower CRP levels
bone metabolism. IFN-c functions both as a pro-resorptive
by Day 30 and Day 90 than calcium supplementation
and an anti-resorptive cytokine ]. IFN-c blocks
RANKL-induced osteoclast differentiation by direct inter-
In summary, R-ELF supplementation reduced osteoclast
action with osteoclast precursors but indirectly stimulates
generation, as indicated by decreased levels of RANKL,
osteoclast formation. Accordingly, the modest 13.5%
and improved inflammatory status of the subjects, as
increase in IFN-c levels observed for the R-ELF group can
indicated by the decline in CRP levels. These changes are
potentially be attributed to this dual property. It is well
attributed to diminished pro-inflammatory and increased
known that pro-inflammatory cytokines IL-6, IL-1b and
anti-inflammatory cytokine levels. Despite being a pre-
TNF-a are increased in post-menopausal estrogen defi-
liminary investigation with a small number of subjects
ciency –In our study, R-ELF supplementation
monitored for a short duration, the results are promising
greatly reduced IL-6 and TNF-a levels compared to
and warrant further investigation by a placebo-controlled
Inflammatory responses in postmenopausal women
study in a larger population. R-ELF, a milk protein-based
evidence for a link between systemic inflammation and osteo-
bone health supplement, could provide a safe and natural
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Doi:10.1016/j.jnoncrysol.2003.08.075
Journal of Non-Crystalline Solids 331 (2003) 217–227 TEM and XRD study of early crystallization of lithium disilicate glasses P.C. Soares Jr. a, E.D. Zanotto a,*, V.M. Fokin a, H. Jain b a LaMaV – Vitreous Materials Laboratory, Universidade Federal de S ao Carlos, 13595-905 S ao Carlos, SP, Brazil b Department of Materials Science and Engineering, Lehigh University, 18015 Bethlehem, PA, USA
biopiraterie.de
Erschienen in: GiD Gen-ethischer Informationsdienst, Nr. 185, Dezember 2007 Bei den Vertragsstaaten der Konvention über die biologische Vielfalt steht derzeit die Entwicklung ei-ner Regulierung der Nutzung genetischer Ressourcen weit oben auf der Agenda. Nichtregierungsor-ganisationen aus sieben Ländern unterstützen mit ihrem Forderungskatalog die Positionen von Netz-werken indigener Gruppen. Die Kritik an der CBD kann jedoch darüber hinaus gehen.