Comparison of bacterial counts in expressed breast milk following standard or strict infection control regimens in neonatal intensive care units: compliance of mothers does matter.

Bacterial counts in 1466 expressed breast milk (EBM) samples from women following one of two infection control regimens (standard vs strict) were investigated. Overall, 12% of samples yielded Gram-negative bacteria, with no significant differences between the standard [11.9% (94/788)] and strict [12.1% (82/678)] regimens (P = 0.92). Significantly more samples were contaminated when expressed at home (standard regimen home/hospital: 17.9% vs 6.1%; strict regimen home/hospital: 19.6% vs 3.4%; P < 0.001). Bacterial contamination of EBM was not associated with the regimen, but was associated with the location of breast milk expression. Attempts to improve personal hygiene during milk collection seem to be of limited value. Good hygiene of collection and storage equipment is likely to be the most important way to ensure the microbiological quality of EBM.

Blood transfusions using 27 gauge PICC lines: a retrospective clinical study on safety and feasibility.

BACKGROUND: Blood transfusions are required by most extremely low birth weight (ELBW) infants, but sometimes an adequate peripheral venous access cannot be achieved. Under these circumstances, we used 27 Gauge (G) peripherally inserted central catheter (PICC) lines that are routinely inserted on the second day of life. Due to their narrow lumen, hemolysis of transfused erythrocytes was a major concern. We therefore performed a retrospective study in ELBW infants to analyze the incidence, safety and feasibility of PRBC transfusions via 27 G PICC lines. METHODS: ELBW infants admitted from 08/2011-07/2012 were screened for packed red blood cell (PRBC) transfusions. Those applied via 27 G PICC lines were identified. For analysis of transfusion safety (hemolysis), hemoglobin and potassium levels as well as cardiovascular variables (invasive mean arterial blood pressure and heart rate) were evaluated before and after transfusion. For analysis of transfusion feasibility, catheter removal after transfusion and the reason for removal were recorded. RESULTS: A total of 648 transfusions were applied in 110 ELBW infants. 27 infants (24%) received no transfusion. In 12/83 (14.5%) infants who received PRBCs, transfusions were applied using a 27 G PICC line (38/648, 5.9%). Patients who received PRBCs via the PICC line were smaller at birth (582 g [range 380-752 g] vs. 710 g [430-972 g]; 23+6 [23+1-27+6] vs. 26+0 [23+1-31+4]) and required a higher number of PRBC transfusions (n=13 vs. n=5) overall. Transfusion analysis showed an appropriate increase of blood hemoglobin levels and stable potassium levels as well as cardiovascular parameters. 4/38 of PICC lines were removed within 24 h after transfusion, one due to occlusion (15 h after transfusion). CONCLUSIONS: We conclude that PRBC transfusions via 27 G PICC lines were feasible and performed without signs of hemolysis in ELBW infants. Our findings may help clinicians in the management of ELBW infants requiring transfusions if a peripheral venous access is not achievable.

Safety of blood transfusions using 27 gauge neonatal PICC lines: an in vitro study on hemolysis.

Blood transfusions are required by the majority of extremely premature infants. Packed red blood cells (PRBCs) are usually applied via simple peripheral cannulas. In situations where no peripheral venous access is achievable, 27 Gauge (G) neonatal PICC lines - that are ideally exclusively dedicated to application of parenteral nutrition - may represent a useful alternative access for PRBC transfusions. However, transfusion via small scaled catheters may damage PRBCs and lead to hemolysis. We here evaluate whether transfusion of irradiated PRBCs via 27 G PICC lines leads to hemolysis in vitro.Experimental transfusions of gamma-irradiated PRBCs were performed at increasing velocities (2.5, 3.7, 5 ml/h; full force manual push approximating 30 ml/h) via 27 G PICC lines of 20 and 30 cm length. Parameters of hemolysis (lactate dehydrogenase, potassium and free hemoglobin) were measured from the supernatants of transfused PRBCs and the percentage of hemolysis was calculated.Potassium and lactate dehydrogenase after transfusion at increasing velocities did not differ significantly from negative controls. Free hemoglobin levels showed a small but significant increase at the slowest transfusion speed (2.5 ml/h) using the 30 cm 27 G PICC line, with a relative hemolysis of only 0.13%. A manual push (approximating 30 ml/h) showed no significant changes of parameters from baseline.We conclude that transfusion of gamma-irradiated PRBCs using a 27 G neonatal PICC line does not cause clinically relevant hemolysis in vitro. Clinical studies are needed to confirm the feasibility and safety of the approach in vivo.

Impact of Doppler sonography on intrauterine management and neonatal outcome in preterm fetuses with intrauterine growth restriction.

OBJECTIVES: To test the hypothesis that earlier delivery may be warranted to improve neonatal outcome of fetuses with intrauterine growth restriction (IUGR) with abnormal Doppler parameters. METHODS: This was a retrospective cohort study of 110 fetuses with an antenatal diagnosis of IUGR due to placental insufficiency which had a very low birth weight (< 1500 g), delivered at the Department of Fetomaternal Medicine of the Medical University of Vienna, Austria, between January 1999 and July 2009. Doppler results before delivery were classified as follows: Group 1: abnormal umbilical artery (UA) pulsatility index (PI) more than 2 SD above the mean for normal reference data, or absent UA end-diastolic flow, both with normal middle cerebral artery (MCA) PI (mean ± 2 SD); Group 2: abnormal UA-PI > mean + 2 SD, or absent or reversed UA end-diastolic flow, with abnormal MCA-PI (< mean - 2 SD) and normal ductus venosus (DV) PI (mean ± 2 SD); Group 3: absent or reversed UA end-diastolic flow, with abnormal MCA-PI (< mean - 2 SD) and abnormal DV-PI (> mean + 2 SD) and/or absent or reversed end-diastolic DV flow. Pregnancy outcome was analyzed according to Doppler results. RESULTS: Due to very poor prognosis, 19 fetuses underwent expectant management and died in utero. These were excluded from further analyses. Of the remaining 91 cases, 17 were in Doppler Group 1, 44 in Group 2 and 30 in Group 3. Within 4 weeks after delivery, 0/17 (0%) infants in Group 1 died, 2/44 (4.5%) infants in Group 2 died and 7/30 (23.3%) infants in Group 3 died (P = 0.019). None of the 42 Group 2 cases that delivered at or after 28 completed gestational weeks died within 4 weeks after delivery, in contrast to 4/20 (20.0%) Group 3 cases (P = 0.009). In comparison, among infants delivered before 27 completed gestational weeks, 2/2 (100%) Group 2 cases died and 3/10 (30.0%) Group 3 cases died; P = 0.152). CONCLUSIONS: Doppler examinations are highly predictive in assessing the outcome of IUGR fetuses. From 28 completed gestational weeks, early delivery before the onset of fetal cardiac decompensation might be beneficial.

Neonatal colonization of mice with Lactobacillus plantarum producing the aeroallergen Bet v 1 biases towards Th1 and T-regulatory responses upon systemic sensitization.

BACKGROUND: The use of recombinant lactic acid bacteria (LAB) as vehicles for mucosal delivery of recombinant allergens is an attractive concept for antigen-defined allergy prevention/treatment. Interventions with LAB are of increasing interest early in life when immune programming is initiated. Here, we investigated the effect of neonatal colonization with a recombinant LAB producing the major birch pollen allergen Bet v 1 in a murine model of type I allergy. METHODS: We constructed a recombinant Lactobacillus (L.) plantarum NCIMB8826 strain constitutively producing Bet v 1 to be used for natural mother-to-offspring mono-colonization of germ-free BALB/c mice. Allergen-specific immunomodulatory effects of the colonization on humoral and cellular immune responses were investigated prior and after sensitization to Bet v 1. RESULTS: Mono-colonization with the Bet v 1 producing L. plantarum induced a Th1-biased immune response at the cellular level, evident in IFN-γ production of splenocytes upon stimulation with Bet v 1. After sensitization with Bet v 1 these mice displayed suppressed IL-4 and IL-5 production in spleen and mesenteric lymph node cell cultures as well as decreased allergen-specific antibody responses (IgG1, IgG2a, and IgE) in sera. This suppression was associated with a significant up-regulation of the regulatory marker Foxp3 at the mRNA level in the spleen cells. CONCLUSION: Intervention at birth with a live recombinant L. plantarum producing a clinically relevant allergen reduces experimental allergy and might therefore become an effective strategy for early intervention against the onset of allergic diseases.

Familial Mediterranean Fever in Crete: a genetic and structural biological approach in a population of 'intermediate risk'.

Familial Mediterranean Fever (FMF) is an autosomal, recessively inherited disease, characterized by recurrent and short attacks of fever with serosal inflammation that are caused by mutations in MEFV gene that encodes pyrin protein. To date, more than 70 disease-associated mutations have been identified, almost all of them representing missense nucleotide changes. FMF is very common among patients with Mediterranean ancestry, although the exact prevalence is not yet known, Greeks are considered to be at 'intermediate risk'. In the present study, we studied FMF patients in natives of Crete, a population sharing a common genetic and cultural background. The spectrum of MEFV gene mutations in 71 patients as well as 158 healthy controls was studied by performing a molecular analysis focused on the 12 most frequent FMF-associated mutations. We found that 59 of 71 (83.1%) FMF patients had at least one MEFV mutation, five patients were homozygotes and 54 heterozygotes for FMF-associated mutations. No mutations were detected in 12 patients (16.9%). As in high-risk populations, common MEFV mutations were found in Cretan FMF patients, with the M694V being the most penetrant. M694V and M694I mutations were associated with severe phenotypes, with many patients presenting with uncommon clinical manifestations such as erysipelas-like erythema or renal disturbances. Of interest, 20 (37%) of our heterozygous FMF patients presented with a severe phenotype. Population genetics analysis showed an FMF carrier frequency in healthy Cretan population of approximately 6% (1:17) and places Cretans closer to the Western rather than Eastern populations of the Mediterranean basin. Finally, we constructed a three-dimensional model showing the interaction of the PRYSPRY domain of pyrin with caspase-1 onto which we mapped MEFV mutations, classified according to disease severity. In this model, the 'flexible loops' of caspase-1 appear to have no access to some positions that have been previously associated with mild disease, suggesting that alternative pathogenic pathways leading to FMF need to be explored.

The European LABDEL project and its relevance to the prevention and treatment of allergies.

In March 2001, the European Commission funded a 3-year project (contract no. QLK3-CT-2000-00340) under the fifth Framework Programme to develop and test prototype products based on the oral delivery of vaccine and therapeutic agents using harmless lactic acid bacteria (LAB). The project, best known under its acronym LABDEL (for LAB delivery) also included research on LAB fermentation and technological innovations aimed at enhancing the efficiency of LAB delivery systems (1). One of the key scientific objectives was to investigate the possibility to prevent or treat a type I allergic disease using mucosal administration of LAB expressing the pollen allergen Bet v 1. The aim of this paper was to describe the background of the project with reference to a limited selection of articles and recent reviews as well as the results and major conclusions arising from this part of the project.

Modulation of allergic immune responses by mucosal application of recombinant lactic acid bacteria producing the major birch pollen allergen Bet v 1.

BACKGROUND: Probiotic lactic acid bacteria (LAB) are able to modulate the host immune system and clinical trials have demonstrated that specific strains have the capacity to reduce allergic symptoms. Therefore, we aimed to evaluate the potential of recombinant LAB producing the major birch pollen allergen Bet v 1 for mucosal vaccination against birch pollen allergy. METHODS: Recombinant Bet v 1-producing Lactobacillus plantarum and Lactococcus lactis strains were constructed. Their immunogenicity was compared with purified Bet v 1 by subcutaneous immunization of mice. Intranasal application of the live recombinant strains was performed to test their immunomodulatory potency in a mouse model of birch pollen allergy. RESULTS: Bet v 1 produced by the LAB was recognized by monoclonal anti-Bet v 1 and IgE antibodies from birch pollen-allergic patients. Systemic immunization with the recombinant strains induced significantly lower IgG1/IgG2a ratios compared with purified Bet v 1. Intranasal pretreatment led to reduced allergen-specific IgE vs enhanced IgG2a levels and reduced interleukin (IL)-5 production of splenocytes in vitro, indicating a shift towards non-allergic T-helper-1 (Th1) responses. Airway inflammation, i.e. eosinophils and IL-5 in lung lavages, was reduced using either Bet v 1-producing or control strains. Allergen-specific secretory IgA responses were enhanced in lungs and intestines after pretreatment with only the Bet v 1-producing strains. CONCLUSIONS: Mucosal vaccination with live recombinant LAB, leading to a shift towards non-allergic immune responses along with enhanced allergen-specific mucosal IgA levels offers a promising approach to prevent systemic and local allergic immune responses.

Serum homocysteine concentration as a marker of nutritional status of healthy subjects in Crete, Greece.

BACKGROUND: Dietary habits are an important determinant of serum homocysteine (tHcy), which may be a marker rather than a cause of progression of the atherosclerotic process. The aim of the present study was to evaluate the nutritional status, and to determine the serum tHcy concentrations in healthy subjects who live in rural areas of Crete, and who theoretically follow a contemporary Mediterranean-style diet. METHODS: Serum tHcy, folate, vitamin B(12), creatinine, glucose, and the lipid profile, were measured in 203 (141 men and 62 women) healthy subjects, aged 33-78 years. The major risk factors for cardiovascular disease such as age, gender, cigarette smoking, obesity were recorded and dietary data were assessed using a 3-day weighed food intake record. RESULTS: Our population had high serum tHcy, low serum folate concentrations and lower than the traditional Cretan dietary folate intake [median (range): 12.0 (3.6-44.7) micromol L(-1), 7.9 (1.9-15.5) ng mL(-1) and 241 (68-1106) microg, respectively]. Dietary intake of fibre, omega-3, and mono- or/ polyunsaturated fatty acids was also low. An inverse relation was observed between serum tHcy concentrations and serum folate (r = -0.28; P < 0.01) and vitamin B(12) levels (r = -0.33; P < 0.001). CONCLUSIONS: Nowadays, the Cretan diet has changed towards a more westernized eating pattern. Given the analytic difficulties in determining the amount of folate in food and the inverse correlation between serum tHcy and folate levels, serum tHcy concentrations may be a useful marker for nutritional status, especially folate deficiency, in healthy subjects.

Influence of the route of sensitization on local and systemic immune responses in a murine model of type I allergy.

The pathophysiological and immunological characteristics of allergic immune responses are controlled by a variety of factors. We have studied the extent to which the route of sensitization influences allergen-specific IgE synthesis and local airway inflammation using a mouse model of allergic sensitization to the major birch pollen allergen Bet v 1. Sensitization of BALB/c mice with recombinant (r)Bet v 1 was performed using intraperitoneal (i.p.), subcutaneous (s.c.) or aerosol (a.s.) sensitization protocols. Mice were analysed for allergen-specific serum antibodies by ELISA and IgE-dependent basophil degranulation. Proliferative responses and cytokine production of splenocytes were measured upon Bet v 1 stimulation in vitro. Bronchoalveolar lavages were performed after airway challenge with aerosolized birch pollen extract for assessment of eosinophilic airway inflammation and local cytokine production in vivo. Highest allergen specific IgE levels and IgE-dependent basophil degranulation were achieved using the SC route. High IL-5 production by spleen and lung cells was associated with pronounced eosinophilia in bronchoalveolar lavages. After i.p. sensitization, despite giving the highest IgG levels, only low IgE levels, basophil degranulation and IL-5 production were seen. On the other hand, a.s. sensitization, resulting in the lowest systemic IgE and IL-5 levels, led to a comparably strong airway inflammation as the s.c. route. Our finding that the route of sensitization can result in a dissociation of local and systemic immune responses may contribute to a better understanding of the pathogenesis of allergic diseases and help to develop new treatment strategies.

Mucosal tolerance as therapy of type I allergy: intranasal application of recombinant Bet v 1, the major birch pollen allergen, leads to the suppression of allergic immune responses and airway inflammation in sensitized mice.

BACKGROUND: Several studies have demonstrated that mucosal administration of soluble antigens can prevent the onset or reduce the severity of certain autoimmune diseases or allergies. Few studies exist showing the efficacy of mucosal tolerance for therapy of such diseases. OBJECTIVE: The aim of the present study was to modulate an allergic immune response by intranasal antigen administration in an already sensitized organism. METHODS: A murine model of allergic asthma to birch pollen (BP) and its major allergen Bet v 1 was utilized. Sensitized mice were intranasally treated with recombinant (r)Bet v 1 in different concentrations and at different intervals. On the day the mice were killed, blood and bronchoalveolar lavage fluids were taken and immediate type I skin tests were performed. T cell proliferation and cytokine production (interleukin (IL)-5, interferon (IFN)-gamma) were measured in spleen and lung cell cultures. RESULTS: Mucosal treatment with rBet v 1 (3 x 50 microg in 4 day intervals) led to a reduction of type I skin reactions, suppressed immunoglobulin (Ig)G1/IgE antibody levels and markedly decreased IL-5 and IFN-gamma production in vitro in spleen and lung cell cultures. Moreover, lung inflammation (i.e. eosinophilia and IL-5 levels in bronchoalveolar lavage fluids) was significantly suppressed by the treatment. CONCLUSION: Our results demonstrate that intranasal treatment with rBet v 1 reduced systemic allergic immune responses as well as airway inflammation in BP-sensitized mice. We therefore suggest that mucosal tolerance induction with recombinant allergens could be a promising concept for the therapy of allergic diseases.

Oligodeoxynucleotides containing CpG motifs modulate the allergic TH2 response of BALB/c mice to Bet v 1, the major birch pollen allergen.

BACKGROUND: The use of adequate adjuvants to modulate the allergic T(H2)-type immune response is a promising concept for future immunotherapy of type I allergy. Bacterial DNA or oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have been demonstrated to foster T(H1)-type immune responses. OBJECTIVE: We investigated the adjuvanticity of CpG-ODNs and their capability to modulate the allergic T(H2) response in a mouse model. METHODS: BALB/c mice were treated with CpG-ODNs and Bet v 1, the major birch pollen allergen, in different experimental setups. Allergen-specific antibody responses, T(H) cytokines, and eosinophilic infiltration of the airways were investigated. RESULTS: Intraperitoneal administration of Bet v 1 together with aluminium hydroxide led to a typical T(H2) response. In contrast, coadminstration of CpG-ODNs with Bet v 1 in aluminium hydroxide resulted in markedly increased T(H1) activities (high IgG2a levels) and subsequently to reduced airway inflammation. The T(H1)-like immune response indicated by these humoral findings was also reflected by decreased IL-5 and increased IFN-gamma levels in cell cultures. CpG-ODNs as sole adjuvants with Bet v 1 did not lead to measureable Ig responses after subcutaneous or intraperitoneal immunizations; after intranasal application, 3 of 10 mice reacted. Nevertheless, a prophylactic effect was obtained with all routes tested; that is, mice treated subsequently with an established aerosol sensitization protocol displayed altered immune responses characterized by drastically elevated levels of Bet v 1-specific IgG2a, indicating a T(H1)/T(H0)-like immunity. Application of CpG-ODNs after aerosol sensitization also induced IgG2a. CONCLUSION: By inducing T(H1)/T(H0)-biased immune responses to allergens, the use of CpG-ODNs as adjuvants may have important impacts for new forms of specific immunotherapy in type I hypersensitivity.

Suppression of antigen-specific T- and B-cell responses by intranasal or oral administration of recombinant bet v 1, the major birch pollen allergen, in a murine model of type I allergy.

BACKGROUND: Mucosal (nasal or oral) administration of soluble protein antigens induces a state of antigen-specific immunologic hyporesponsiveness. Several studies have shown that induction of mucosal tolerance can prevent the onset or reduce the severity of certain TH1 -mediated experimental autoimmune diseases. Only a few studies describe similar results for type I allergies, which are caused by excessive TH2 cell activities. OBJECTIVE: We sought to investigate whether mucosal tolerance induction would also be efficient in preventing an allergic type I immune response. METHODS: A murine model of inhalative type I allergy, leading to sensitization to birch pollen and its major allergen Bet v 1 in BALB/c mice, was used. Recombinant Bet v 1 was nasally or orally applied in low doses before sensitization. At the time of death, immediate-type skin tests were performed. Blood was taken, and serum was used for measurement of allergen-specific antibodies. Spleen cell cultures were performed to determine cytokine production (IL-4, IL-5, IL-10, and IFN-gamma), as well as levels of TGF-beta mRNA. RESULTS: Both nasal and oral administration of minimal doses of recombinant Bet v 1 before aerosol sensitization with birch pollen suppressed the allergen-specific antibody production of all isotypes. Consequently, the in vivo type I skin test responses to the allergen were negative in the tolerized, in contrast to the sensitized, group. Moreover, allergen-specific lymphoproliferative responses and cytokine production in vitro (ie, IFN-gamma, IL-4, IL-5, and IL-10) were markedly reduced. In contrast, expression of TGF-beta mRNA was markedly increased in spleen cells from nasally tolerized animals, indicating regulatory mechanisms for tolerance induction. CONCLUSION: We conclude from the present study that nasal, as well as oral, administration of recombinant allergen is an effective way to prevent allergen-specific T- and B-cell responses in a TH2 model.

Modulation of an allergic immune response via the mucosal route in a murine model of inhalative type-I allergy.

A murine model of aerosol inhalation, leading to sensitization to birch pollen (BP) and its major allergen Bet v 1, was established in order to try to influence type-I allergic immune responses via the mucosal route. We previously demonstrated that simultaneous inhalation of BP and cholera toxin, a potent mucosal adjuvant, induced a Th1-like immune response to the allergen in naive mice and modulated allergic immune responses in sensitized mice. In contrast to cholera holotoxin, mucosal application of the cholera B subunit (CTB) conjugated to antigen has been shown to induce peripheral tolerance in certain models of Th1-based autoimmune diseases. In the present study we investigated the potential of such an antigen delivery system to suppress Th2-based, allergic immune responses. Mucosal administration of CTB/Bet v 1 conjugates prior to sensitization led to significantly increased allergen-specific IgE/IgG1 and IgG2a antibody levels and cytokine production (IL-5, IFN-gamma) in vitro. Thus, CTB coupled to Bet v 1 acted as an adjuvant rather than a tolerogen. On the other hand we noted that mucosal application of CTB coupled to ovalbumin led to marked suppression of antigen-specific IgE antibody levels and IL-5 production in vitro and thereby restricted allergic sensitization. These results indicated that the effects of CTB/antigen conjugates depended on the nature of the antigen. In contrast to Bet v 1 coupled to CTB, nasal as well as oral application of low doses of unconjugated, Bet v 1 prior to aerosol sensitization inhibited allergen-specific antibody responses of all isotypes, cutaneous type-I skin tests in vivo as well as allergen-specific lymphoproliferative responses and cytokine production (IL-4, IL-5, IL-10, IFN-gamma) in vitro, suggesting that both T- and B-cell tolerance to the allergen were induced. Taken together, mucosal tolerance induction as well as the use of certain transmucosal antigen delivery systems might be promising new strategies to modulate type-I allergic immune responses