Short primiparous women are at an increased risk for gestational diabetes mellitus.

OBJECTIVES: Both short stature and adiposity are risk factors for gestational diabetes mellitus (GDM). The aim of this study was to simultaneously evaluate the importance of stature and degree of adiposity on development of GDM in primiparous women. STUDY DESIGN: Longitudinal cohort study. METHODS: In the city of Vantaa, Finland, between 2009 and 2015, all together 7750 primiparous women without previously diagnosed diabetes mellitus gave birth. Of these, 5223 women were ≥18 years of age with information on height, weight, and complete data from a 75 g 2-h oral glucose tolerance test composing the study participants of this study. RESULTS: A 155-cm tall woman with a body mass index (BMI) of 25.5 kg/m2 had a similar risk for GDM as a 175-cm tall woman with a BMI of 27.1 kg/m2. Women shorter than 159 cm had the highest prevalence of GDM, 28.7%, whereas women with height between 164 and 167 cm had the lowest prevalence of GDM, 19.9% (P < 0.001). Height was inversely and significantly associated with both 1- and 2-h glucose values (both P < 0.001). CONCLUSIONS: To avoid over diagnosis of GDM, an unbiased strategy is needed to determine and diagnose GDM in women with different stature and degree of adiposity.

Public health response to a measles outbreak on a university campus in Australia, 2015.

This report describes the effective public health response to a measles outbreak involving a university campus in Brisbane, Australia. Eleven cases in total were notified, mostly university students. The public health response included targeted measles vaccination clinics which were established on campus and focused on student groups most likely to have been exposed. The size of the university population, social interaction between students on and off campus, as well as limited vaccination records for the university community presented challenges for the control of this extremely infectious illness. We recommend domestic students ensure vaccinations are current prior to matriculation. Immunisation information should be included in university student enrolment packs. Incoming international students should ensure routine vaccinations are up-to-date prior to arrival in Australia, thereby reducing the risk of importation of measles and other infectious diseases.

Breast milk IgA to foods has different epitope specificity than serum IgA-Evidence for entero-mammary link for food-specific IgA?

BACKGROUND: We have previously shown that maternal cow's milk (CM) elimination results in downregulation of CM-specific IgA antibody levels in BM, but not in serum, suggesting that an entero-mammary link may exist for food-specific antibody-secreting cells. OBJECTIVE: We sought to investigate whether food-specific IgA epitope profiles differ intra-individually between mother's serum and BM. We also examined how infants' food epitope-specific IgA develops in early infancy and the relationship of IgA epitope recognition with development of cow's milk allergy (CMA). METHODS: We measured specific IgA to a series of overlapping peptides in major CM allergens (αs1 -, αs2 -, ß- and κ-caseins and ß-lactoglobulin) in paired maternal and infant serum as well as BM samples in 31 mother-infant dyads within the first 15 post-partum months utilizing peptide microarray. RESULTS: There was significant discordance in epitope specificity between BM and maternal sera ranging from only 13% of sample pairs sharing at least one epitope in αs1 -casein to 73% in κ-casein. Epitope-specific IgA was detectable in infants' sera starting at less than 3 months of age. Sera of mothers with a CMA infant had increased binding of epitope-specific IgA to CM proteins compared to those with a non-CMA infant. CONCLUSION & CLINICAL RELEVANCE: These findings support the concept that mother's milk has a distinct antifood antibody repertoire when compared to the antibody repertoire of the peripheral blood. Increased binding of serum epitope-specific IgA to CM in mothers of infants with CMA may reflect inherited systemic immunogenicity of CM proteins in these families, although specific IgA in breast milk was not proportionally up-regulated.

Role of maternal elimination diets and human milk IgA in the development of cow's milk allergy in the infants.

BACKGROUND: The role of maternal avoidance diets in the prevention of food allergies is currently under debate. Little is known regarding the effects of such diets on human milk (HM) composition or induction of infant humoral responses. OBJECTIVE: To assess the association of maternal cow's milk (CM) avoidance during breastfeeding with specific IgA levels in HM and development of cow's milk allergy (CMA) in infants. METHODS: We utilized HM and infant serum samples from a prospective birth cohort of 145 dyads. Maternal serum and HM samples were assessed for casein and beta-lactoglobulin (BLG)-specific IgA and IgG by ELISA; 21 mothers prophylactically initiated a strict maternal CM avoidance diet due to a sibling's history of food allergy and 16 due to atopic eczema or regurgitation/vomiting seen in their infants within the first 3 months of life. Infants' sera were assessed for casein and BLG-specific IgG, IgA and IgE; CMA was confirmed by an oral food challenge. The impact of HM on BLG uptake was assessed in transcytosis assays utilizing Caco-2 intestinal epithelial cell line. RESULTS: Mothers avoiding CM had lower casein- and BLG-specific IgA in HM than mothers with no CM restriction (P = 0.019 and P = 0.047). Their infants had lower serum casein- and BLG-specific IgG(1) (P = 0.025 and P < 0.001) and BLG-specific IgG(4) levels (P = 0.037), and their casein- and BLG-specific IgA levels were less often detectable than those with no CM elimination diet (P = 0.003 and P = 0.007). Lower CM-specific IgG4 and IgA levels in turn were associated with infant CMA. Transcytosis of BLG was impaired by HM with high, but not low levels of specific IgA. CONCLUSIONS: Maternal CM avoidance was associated with lower levels of mucosal-specific IgA levels and the development of CMA in infants. CLINICAL RELEVANCE: HM IgA may play a role in preventing excessive, uncontrolled food antigen uptake in the gut lumen and thereby in the prevention of CMA.

Presence of functional, autoreactive human milk-specific IgE in infants with cow's milk allergy.

BACKGROUND: Occasionally, exclusively breastfed infants with cow's milk allergy (CMA) remain symptomatic despite strict maternal milk avoidance. OBJECTIVE: To determine whether or not persistence of symptoms could be due to sensitization against endogenous human milk proteins with a high degree of similarity to bovine allergens. METHODS: Ten peptides representing known bovine milk IgE-binding epitopes [α-lactalbumin (ALA), ß- and κ-casein] and the corresponding, highly homologous human milk peptides were labelled with sera from 15 breastfed infants with CMA, aged 3 weeks to 12 months, and peptide (epitope)-specific IgE antibodies were assessed. Nine of the 15 breastfed infants became asymptomatic during strict maternal avoidance of milk and other major food allergens; six infants remained symptomatic until weaned. Ten older children, aged 5-15 years, with CMA were also assessed. The functional capacity of specific IgE antibodies was assessed by measuring ß-hexosaminidase release from rat basophilic leukaemia cells passively sensitized and stimulated with human and bovine ALA. RESULTS: A minimum of one human milk peptide was recognized by IgE antibodies from 9 of 15 (60%) milk-allergic infants, and the majority of older children with CMA. Genuine sensitization to human milk peptides in the absence of IgE to bovine milk was occasionally seen. There was a trend towards specific IgE being detected to more human milk peptides in those infants who did not respond to the maternal milk elimination diet than in those who did (P = 0.099). Functional IgE antibody to human ALA was only detected in infants not responding to the maternal diet. CONCLUSIONS AND CLINICAL RELEVANCE: Endogenous human milk epitopes are recognized by specific IgE from the majority of infants and children with CMA. Such autoreactive, human milk-specific IgE antibodies appear to have functional properties in vitro. Their role in provoking allergic symptoms in infants exclusively breastfed by mothers strictly avoiding dietary milk remains unclear.

Biocompatible photocrosslinked poly(ester anhydride) based on functionalized poly(epsilon-caprolactone) prepolymer shows surface erosion controlled drug release in vitro and in vivo.

Star-shaped poly(epsilon-caprolactone) oligomers functionalized with succinic anhydride were used as prepolymers to prepare photocrosslinked poly(ester anhydride) to evaluate their in vivo drug delivery functionality and biocompatibility. Thus, in this work, erosion, drug release and safety of the photocrosslinked poly(ester anhydride) were examined in vitro and in vivo. A small water-soluble drug, propranolol HCl (M(w) 296 g/mol, solubility 50 mg/ml), was used as the model drug in an evaluation of the erosion controlled release. Drug-free and drug-loaded (10-60% w/w) poly(ester anhydride) discoids eroded in vitro (pH 7.4 buffer, +37 degrees C) linearly within 24-48 h. A strong correlation between the polymer erosion and the linear drug release in vitro was observed, indicating that the release had been controlled by the erosion of the polymer. Similarly, in vivo studies (s.c. implantation of discoids in rats) indicated that surface erosion controlled drug release from the discoids (drug loading 40% w/w). Oligomers did not decrease cell viability in vitro and the implanted discoids (s.c., rats) did not evoke any cytokine activity in vivo. In summary, surface erosion controlled drug release and the safety of photocrosslinked poly(ester anhydride) were demonstrated in this study.

In vivo delivery of a peptide, ghrelin antagonist, with mesoporous silicon microparticles.

Peptides may represent potential treatment options for many severe illnesses. However, they need an effective delivery system to overcome rapid degradation after their administration. One possible way to prolong peptide action is to use particulate drug delivery systems. In the present study, thermally hydrocarbonized mesoporous silicon (THCPSi) microparticles (38-53 microm) were studied as a peptide delivery system in vivo. D-lys-GHRP6 (ghrelin antagonist, GhA) was used as a model peptide. The effects of GhA-loaded THCPSi microparticles on food intake (s.c., GhA dose 14 mg/kg) and on blood pressure (s.c., GhA dose 4 mg/kg) were examined in mice and rats, respectively. In addition, the effects of THCPSi microparticles (2 mg) on cytokine secretion in mice after single s.c. administration were examined by determining several cytokine plasma concentrations. The present results demonstrate that GhA can be loaded into THCPSi microparticles with a high loading degree (20% w/w). GhA loaded THCPSi microparticles inhibited food intake for a prolonged time, and increased blood pressure more slowly than encountered with a GhA solution. Furthermore, THCPSi microparticles did not increase cytokine activity. The present results suggest that THCPSi might be used as a drug delivery system for peptides.

Selective iNOS inhibitor 1400W enhances anti-catabolic IL-10 and reduces destructive MMP-10 in OA cartilage. Survey of the effects of 1400W on inflammatory mediators produced by OA cartilage as detected by protein antibody array.

OBJECTIVES: In osteoarthritis (OA), the balance between catabolic and anabolic mediators and their regulators in cartilage is disturbed. Proinflammatory cytokine interleukin-1 (IL-1) plays a central role in cartilage destruction and nitric oxide (NO) mediates many of its destructive effects. In the present study, we investigated the secretion of 40 mediators related to inflammation or cartilage degradation by OA cartilage samples with a protein antibody array. The effects of IL-1 and a selective iNOS-inhibitor 1400W on the mediator release were also studied. METHODS: Cartilage tissue was obtained from the leftover pieces of total knee replacement surgery from OA patients. Protein antibody array was used to measure production of 40 mediators in the culture medium. ELISA was used to confirm the antibody array results. RESULTS: OA cartilage secreted spontaneously 15 out of the 40 measured mediators. IL-1Beta enhanced production of 11 of these inflammatory mediators in OA cartilage along with increased NO production. Treatment with a selective iNOS inhibitor 1400W enhanced the production of IL-10, while the levels of MMP-10 were reduced in IL-1 -treated OA cartilage. CONCLUSION: OA cartilage produces many of the mediators involved in the pathogenesis of OA. The ability of 1400W to enhance levels of anti-catabolic IL-10 and to reduce levels of destructive MMP-10 points to the anti-inflammatory mechanisms that iNOS-inhibitors may have.

Identification of amino acids critical for IgE-binding to sequential epitopes of bovine kappa-casein and the similarity of these epitopes to the corresponding human kappa-casein sequence.

BACKGROUND: The delineation of allergenic (i.e. IgE-binding) epitopes in cow's milk proteins and the amino acids (AAs) critical for IgE-binding is necessary to understand better the structural properties of an allergen and to develop more efficacious immunotherapeutic reagents. Furthermore, this information may enable us to understand better cross-sensitivity between different allergens. METHODS: Eleven peptides, 10-14 AAs in length, representing the IgE-binding epitopes of kappa-casein were synthesized on a derivatized cellulose membrane with single AA substitutions at each position. Membranes were incubated with pooled sera from 15 milk-allergic patients and individual sera from 10 of the patients included in the pool. RESULTS: For 10/11 allergenic peptides, one to five different single AA substitutions resulted in elimination of IgE-binding of pooled patient sera. Overall at least one mutated peptide could be found for these 10 IgE-binding sites that resulted in a reduction of IgE-binding in at least 80% of the patients who recognized the native protein. Furthermore, the IgE-binding region at AA104-112 on bovine kappa-casein showed a high degree of similarity with the human kappa-casein, respectively, including the AAs critical for IgE-binding. CONCLUSION: This finding suggests that critical AAs should be assessed with both pooled and individual patient sera to account for the B-cell epitope heterogeneity between patients, with cow's milk allergy. In addition, we identified two potentially cross-reactive peptides between bovine and human caseins of unknown clinical relevance.

Specificity of IgE antibodies to sequential epitopes of hen's egg ovomucoid as a marker for persistence of egg allergy.

BACKGROUND: Approximately two-thirds of egg-allergic infants become tolerant within the first 5 years of life. OBJECTIVE: We sought (1) to compare the recognition of sequential (linear) and conformational binding sites of ovomucoid, ovalbumin and ovotransferrin, by IgE antibodies of children with persistent and transient egg allergy, (2) to identify immunodominant IgE-and IgG-binding epitopes of ovomucoid, and (3) to compare epitope-specificity of IgE antibodies between patients with differing natural histories of egg allergy. METHODS: Using immunodot-blots or ImmunoCAPs, IgE-antibodies against conformational (native) and sequential (reduced and alkylated) egg proteins were determined at the time of clinical reactivity in patients who retained their allergy and in those who developed clinical tolerance. IgE- and IgG-binding epitopes were mapped for ovomucoid using overlapping decapeptides on SPOTs membranes. Recognition of the major IgE-binding epitopes were compared between patients with differing natural histories of egg allergy. RESULTS: The patients with long-lasting egg allergy had a higher concentrations of IgE antibodies against sequential and native ovomucoid and ovalbumin than the children who subsequently gained tolerance (P < 0.01). Four major IgE-binding epitopes were identified in ovomucoid at amino acid 1-10, 9-20, 47-56, and 113-124. IgE antibodies of all seven patients with persistent egg allergy recognized these epitopes whereas none of the 11 children who outgrew their egg allergy did so. CONCLUSIONS: Patients with persistent egg allergy develop IgE antibodies against more sequential and conformational epitopes of ovomucoid and ovalbumin. The presence of serum IgE antibodies to specific sequential epitopes of ovomucoid may be used as a screening instrument for persistent egg allergy.

Mutational analysis of immunoglobulin E-binding epitopes of beta-casein and beta-lactoglobulin showed a heterogeneous pattern of critical amino acids between individual patients and pooled sera.

BACKGROUND: For immunotherapeutic approaches, 'critical' amino acids (AAs) within allergenic epitopes are replaced with alternate AAs to eliminate IgE antibody binding. OBJECTIVE: To determine the critical AAs for IgE binding in beta-casein and beta-lactoglobulin (BLG). METHODS: Peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single AA substitutions (alanine or glycine) at each position. Membranes were incubated with a pool of sera from 15 cow's milk-allergic patients and individual sera from six of the 15 patients. In cases where no decrease in binding occurred with a single AA substitution, peptides with two AA substitutions were generated and labelled. RESULTS: Using pooled patient sera, single AA substitutions led to complete elimination of binding to six of 11 peptides for beta-casein and to all six peptides for BLG. Substituting two AAs led to an elimination of binding to four of the remaining five beta-casein epitopes. However, in three of the 11 modified beta-casein peptides and five of the six BLG peptides, no decrease in IgE binding occurred in at least one individual patient. For these patients, critical AAs other than those defined by the patient serum pool were identified, indicating a heterogeneous pattern of IgE recognition. CONCLUSION: These results indicate that AAs critical for IgE binding are more heterogeneous than initially defined by pooled milk-allergic patient sera. For future immunotherapeutic interventions with mutated peptides, critical AAs should also be identified with individual patient sera to account for heterogeneity of IgE binding between patients.

In vitro evaluation of the effect of cyclodextrin complexation on pulmonary deposition of a peptide, cyclosporin A.

The effect of hydroxypropyl-alpha-cyclodextrin (HP-alpha-CD) complexation on in vitro pulmonary deposition of a cyclic peptide cyclosporin A (CsA) was studied. In addition, the effect of storage (32 days, 40 degrees C, 75% RH) on CsA/HP-alpha-CD complexes was studied. The complexation of CsA with CDs was evaluated by a phase-solubility method. Solid CsA/HP-alpha-CD complexes were prepared by freeze drying. Three inhalation formulations were prepared: CsA/lactose reference formulation (LF) (drug:carrier 1:364, w/w), CsA/HP-alpha-CD complex formulation (CDF) (drug:CD 1:269, w/w) and CsA/HP-alpha-CD complex/lactose formulation (CDLF) (complex:carrier 100:114, w/w). The inhalation studies were performed in vitro using Andersen Sampler (Ph. Eur.) and Taifun multi-dose dry powder inhalers (DPIs). Before the storage, the respirable fraction value (RF%) of CsA was 19.8+/-0.7%, 33.0+/-7.0% and 34.6+/-1.1% (mean+/-S.D., n=4 x 20) with LF, CDF and CDLF, respectively. When exposed to moisture (storage in a permeable polystyrene tube), the RF% values of CsA from formulations containing CsA/HP-alpha-CD complexes were lower than before the storage. However, when stored in the Taifun DPI, the RF% value of CsA from any of the formulations did not decrease. In conclusion, an acceptable RF% value of a peptide CsA from freeze-dried, simply micronized CsA/HP-alpha-CD complex powder was achieved before and after storage in the DPI.

Drug release from starch-acetate microparticles and films with and without incorporated alpha-amylase.

Acetylation of starch considerably decreases its swelling and enzymatic degradation. Thus, starch-acetate (SA) based delivery systems may be suitable for controlled drug delivery. The aim of the present study was to evaluate drug release from the SA microparticles (SA mps) and SA films. The average degree of acetyl substitution (DS) per glucose residue in the starch was either 1.9 (SA DS 1.9) or 2.6 (SA DS 2.6). Timolol (mw 332), calcein (mw 623) and bovine serum albumin (BSA, mw 68,000) were used as model drugs. A continuous timolol release from the both SA mps was observed in phosphate buffer solution (PBS) pH 7.4 (50-days incubation). The release of timolol was faster from the SA DS 1.9 mps than from the SA DS 2.6 mps. Calcein release from both SA mps was continuous in PBS pH 7.4 (5-days incubation). But, calcein release profile from the SA DS 2.6 film in PBS pH 7.4 showed discontinuities. However, the release of calcein from both SA films was continuous in human serum in vitro during the 7-day incubation, i.e. enzymes enhanced calcein release. Thus, alpha-amylase was incorporated into the SA films in order to enhance drug release from the films. However, the effects of incorporation of alpha-amylase on the model macromolecule (BSA) release from the SA films were modest. In conclusion, this study demonstrates the achievement of slow release of different molecular weight model drugs from the SA mps and films as compared to fast release from the native starch preparations. DS of SA, physicochemical properties of a drug and the presence of enzymes can all affect drug release profiles from SA based preparations.

Expression of intercellular adhesion molecules on circulating lymphocytes in relation to different manifestations of cow's milk allergy.

BACKGROUND: The complex interactions between immune cells are partly mediated by different adhesion molecules, but little is known about their role in the systemic immunoinflammatory process following sensitization to food antigens in early infancy. OBJECTIVE: The aim of this study was to investigate the expression of intercellular adhesion molecule-1 (ICAM-1or CD54) and the alpha subunits of its ligands' lymphocyte function-associated antigen-1 (LFA-1) (alphaL subunit or CD11a) and Mac-1 (alphaM subunit or CD11b) on peripheral blood leucocytes in infants with cow's milk allergy (CMA) and in healthy controls. METHODS: Thirty-nine breastfed infants, aged from 0.6 to 8.3 months, and their lactating mothers were included in the study from delivery onwards. During follow-up, 25 infants developed CMA and 14 remained healthy. Expressions of CD54 and CD11b on peripheral blood leucocytes were evaluated by flow cytometry. In addition, the expression of CD11a on peripheral blood leucocytes was analysed by immunocytochemistry. Mothers' milk samples were collected and their leucocyte content was evaluated using a light microscope. RESULTS: The frequency of ICAM-1 expressing peripheral blood lymphocytes was significantly higher in patients with CMA than in healthy infants (P=0.03, Mann-Whitney U-test). Furthermore, the high proportion of ICAM-1-expressing cells was associated with gastrointestinal and multiorgan symptoms in the CMA infants. There was no significant difference in the expression of Mac-1 alphaM on lymphocytes in our study groups, but the LFA-1 alphaL expression seemed to be higher in the IgE-mediated CMA. CONCLUSION: We suggest that the high expression of ICAM-1 on peripheral blood lymphocytes may reflect enhanced stimulation of T cells in vivo and their migration to the effector tissues in an early-phase of developing CMA. Furthermore, high ICAM-1 expression may be associated with the presence of multiorgan manifestations of CMA, whereas high LFA-1 expression may reflect the IgE-mediated disease.

Concurrent cereal allergy in children with cow's milk allergy manifested with atopic dermatitis.

BACKGROUND: There is increasing consensus about the significance of food allergens in the pathogenesis of atopic dermatitis (AD) in infancy and childhood, with cow's milk and egg accounting for most of the reactions. Previous studies have indicated that multiple food sensitization, such as cereals, is very common in patients with cow's milk allergy (CMA). Evidence is lacking, however, as to its clinical relevance. OBJECTIVE: The purpose of this study was to determine the concurrent occurrence of cereal allergy among children with challenge-proven CMA who have residual symptoms, such as AD and/or gastrointestinal symptoms, during cow's milk elimination diet. Further, we sought to evaluate the utility of patch testing in prescreening foods other than cow's milk behind allergic symptoms in children. METHODS: The study population comprised 90 children, aged from 2.5 to 36 months (mean 1.1 years), with challenge-proven CMA. As a result of residual symptoms during meticulous cow's milk elimination diet (AD: n=80, and gastrointestinal: n=10), the children were put on a cereal elimination diet (oats, wheat, rye, and barley) and skin prick tests (SPT) and patch testing with cereals were performed. Open cereal challenge was performed to confirm cereal allergy. RESULTS: Cereal challenge was positive in 66 (73%) of the children with CMA. Of them, 17% reacted with immediate reactions and delayed-onset reactions were seen in 83% of the children. SPT was positive in 23%, patch test in 67%, and either SPT or patch test was positive in 73% of the children with cereal allergy. SPT gave the best positive predictive value, whereas SPT together with patch test gave the best negative predictive value. CONCLUSIONS: Residual symptoms, such as eczema or gastrointestinal symptoms in CMA children may be a sign of undetected allergy to other food antigens. SPT with cereals aids in diagnosing cereal allergy in small children, especially when used together with patch testing.

IgE and IgG binding epitopes on alpha-lactalbumin and beta-lactoglobulin in cow's milk allergy.

BACKGROUND: Cow's milk is one of the most common causes of food allergy in the first years of life. We recently defined IgE and IgG binding epitopes for alpha(s1)-casein, a major cow's milk allergen, and found an association between recognition of certain epitopes and clinical symptoms of cow's milk allergy (CMA). Since alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) are suspected to be significant allergens in cow's milk, we sought to determine the structure of sequential epitopes recognized by IgE antibodies to these proteins. We further sought to assess the pattern of epitope recognition in association with the clinical outcome of CMA. METHODS: According to the known amino acid sequence of ALA and BLG, 57 and 77 overlapping decapeptides (offset by two amino acids), respectively, were synthesized on a cellulose derivatized membrane. Sera from 11 patients 4-18 years of age with persistent CMA (IgE to cow's milk >100 kU(A)/l) were used to identify IgE binding epitopes. In addition, 8 patients < 3 years of age and likely to outgrow their milk allergy (IgE to cow's milk < 30 kU(A)/l) were used to investigate the differences in epitope recognition between patients with 'persistent' and those with 'transient' CMA. Seven patients 4-18 years of age were used for assessing the IgG binding regions. RESULTS: In patients with persistent allergy, four IgE binding and three IgG binding regions were identified on ALA, and seven IgE and six IgG binding epitopes were detected on BLG. The younger patients that are likely to outgrow their allergy recognized only three of these IgE binding epitopes on BLG and none on ALA. CONCLUSIONS: The presence of IgE antibodies to multiple linear allergenic epitopes may be a marker of persistent CMA. The usefulness of IgE binding to distinct epitopes on whey proteins in defining the patients that would have a lifelong CMA needs to be investigated in further studies.