Human CD40 ligand deficiency dysregulates the macrophage transcriptome causing functional defects that are improved by exogenous IFN-γ.

BACKGROUND: CD40 ligand (CD40L) deficiency predisposes to opportunistic infections, including those caused by fungi and intracellular bacteria. Studies of CD40L-deficient patients reveal the critical role of CD40L-CD40 interaction for the function of T, B, and dendritic cells. However, the consequences of CD40L deficiency on macrophage function remain to be investigated. OBJECTIVES: We sought to determine the effect of CD40L absence on monocyte-derived macrophage responses. METHODS: After observing the improvement of refractory disseminated mycobacterial infection in a CD40L-deficient patient by recombinant human IFN-γ (rhIFN-γ) adjuvant therapy, we investigated macrophage functions from CD40L-deficient patients. We analyzed the killing activity, oxidative burst, cytokine production, and in vitro effects of rhIFN-γ and soluble CD40 ligand (sCD40L) treatment on macrophages. In addition, the effect of CD40L absence on the macrophage transcriptome before and after rhIFN-γ treatment was studied. RESULTS: Macrophages from CD40L-deficient patients exhibited defective fungicidal activity and reduced oxidative burst, both of which improved in the presence of rhIFN-γ but not sCD40L. In contrast, rhIFN-γ and sCD40L ameliorate impaired production of inflammatory cytokines. Furthermore, rhIFN-γ reversed defective control of Mycobacterium tuberculosis proliferation by patients' macrophages. The absence of CD40L dysregulated the macrophage transcriptome, which was improved by rhIFN-γ. Additionally, rhIFN-γ increased expression levels of pattern recognition receptors, such as Toll-like receptors 1 and 2, dectin 1, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin in macrophages from both control subjects and patients. CONCLUSION: Absence of CD40L impairs macrophage development and function. In addition, the improvement of macrophage immune responses by IFN-γ suggests this cytokine as a potential therapeutic option for patients with CD40L deficiency.

Paracoccidioidomycosis Associated With a Heterozygous STAT4 Mutation and Impaired IFN-γ Immunity.

Background: Mutations in genes affecting interferon-γ (IFN-γ) immunity have contributed to understand the role of IFN-γ in protection against intracellular pathogens. However, inborn errors in STAT4, which controls interleukin-12 (IL-12) responses, have not yet been reported. Our objective was to determine the genetic defect in a family with a history of paracoccidioidomycosis. Methods: Genetic analysis was performed by whole-exome sequencing and Sanger sequencing. STAT4 phosphorylation (pSTAT4) and translocation to the nucleus, IFN-γ release by patient lymphocytes, and microbicidal activity of patient monocytes/macrophages were assessed. The effect on STAT4 function was evaluated by site-directed mutagenesis using a lymphoblastoid B cell line (B-LCL) and U3A cells. Results: A heterozygous missense mutation, c.1952 A>T (p.E651V) in STAT4 was identified in the index patient and her father. Patient's and father's lymphocytes showed reduced pSTAT4, nuclear translocation, and impaired IFN-γ production. Mutant B-LCL and U3A cells also displayed reduced pSTAT4. Patient's and father's peripheral blood mononuclear cells and macrophages demonstrated impaired fungicidal activity compared with those from healthy controls that improved in the presence of recombinant human IFN-γ, but not rhIL-12. Conclusion: Our data suggest autosomal dominant STAT4 deficiency as a novel inborn error of IL-12-dependent IFN-γ immunity associated with susceptibility to paracoccidioidomycosis.

Dendritic cells from X-linked hyper-IgM patients present impaired responses to Candida albicans and Paracoccidioides brasiliensis.

BACKGROUND: Patients with X-linked hyper-IgM syndrome (X-HIGM) due to CD40 ligand (CD40L) mutations are susceptible to fungal pathogens; however, the underlying susceptibility mechanisms remain poorly understood. OBJECTIVE: To determine whether monocyte-derived dendritic cells (DCs) from patients with X-HIGM exhibit normal responses to fungal pathogens. METHODS: DCs from patients and controls were evaluated for the expression of costimulatory (CD80 and CD86) and MHC class II molecules and for their ability to produce IL-12 and IL-10 in response to Candida albicans and Paracoccidioides brasiliensis. We also evaluated the ability of C albicans- and P brasiliensis-pulsed mature DCs to induce autologous T-cell proliferation, generation of T helper (T(H)) 17 cells, and production of IFN-γ, TGF-ß, IL-4, IL-5, and IL-17. RESULTS: Immature DCs from patients with X-HIGM showed reduced expression of CD80, CD86, and HLA-DR, which could be reversed by exogenous trimeric soluble CD40L. Most important, mature DCs from patients with X-HIGM differentiated by coculturing DCs with fungi secreted minimal amounts of IL-12 but substantial amounts of IL-10 compared with mature DCs from normal individuals. Coculture of mature DCs from X-HIGM patients with autologous T cells led to low IFN-γ production, whereas IL-4 and IL-5 production was increased. T-cell proliferation and IL-17 secretion were normal. Finally, in vitro incubation with soluble CD40L reversed the decreased IL-12 production and the skewed T(H)2 pattern response. CONCLUSION: Absence of CD40L during monocyte/DC differentiation leads to functional DC abnormalities, which may contribute to the susceptibility to fungal infections in patients with X-HIGM.

Probiotic supplementation in marathonists and its impact on lymphocyte population and function after a marathon: a randomized placebo-controlled double-blind study.

Probiotic supplementation arises as playing an immune-stimulatory role. High-intensity and -volume exercise can inhibit immune cell function, which threatens athletic performance and recovery. We hypothesized that 30 days of probiotic supplementation could stabilize the immune system of athletes preventing immune suppression after a marathon race. Twenty-seven male marathonists were double-blinded randomly into probiotic (Bifidobacterium-animalis-subsp.-Lactis (10 × 109) and Lactobacillus-Acidophilus (10 × 109) + 5 g of maltodextrin) and placebo (5 g of maltodextrin) group. They received 30 sachets and supplemented 1 portion/day during 30 days before the race. Blood were collected 30 days before (rest), 1 day before (pre), 1 h after (post) and 5 days after the race (recovery). Both chronic and acute exercise modulated a different T lymphocyte population (CD3+CD4-CD8- T-cells), increasing pre-race, decreasing post and returning to rest values at the recovery. The total number of CD8 T cell and the memory subsets statistically decreased only in the placebo group post-race. Pro-inflammatory cytokine production by stimulated lymphocytes decreased in the probiotic group after the supplementation period. 30 days of probiotic supplementation maintained CD8 T cell and effector memory cell population and played an immunomodulatory role in stimulated lymphocytes. Both, training and marathon modulated a non-classical lymphocyte population regardless of probiotic supplementation.

Transfer of antibodies across the placenta and in breast milk from mothers on intravenous immunoglobulin.

We studied the levels of immunoglobulins in colostrum, milk and sera from two common variable immunodeficiency (CVID) mothers (M1 and M2), and in sera from their newborn infants. During pregnancy they continued intravenous immunoglobulin therapy (IVIG). Antibody levels from maternal and cord blood collected at delivery and colostrum and milk, collected on the 3rd and 7th post-partum days, respectively, were analyzed. Although cord/maternal blood ratios of total immunoglobulins and subclasses, as well as specific antibodies differed between M1 and M2, both showed good placental transfer of anti-protein and anti-polysaccharide antibodies, despite lower cord/maternal blood ratios in M2. Anti-Streptococcus pneumoniae antibody avidity indexes were similar between paired maternal and cord serum. Both mothers' colostrum and milk samples showed only traces of IgA, and IgM and IgG levels in colostrum were within normal range in M1, whereas M2 presented elevated IgG and low IgM levels, when compared with healthy mothers. The study of colostrum and milk activity showed that they strongly inhibited enteropathogenic Escherichia coli adhesion in vitro. CVID patients must be informed about the relevance of regular IVIG administration during pregnancy, not only for their own health but also for their immune immature offspring. Breast-feeding should be encouraged as colostra from these CVID patients strongly inhibited E. coli adhesion to human epithelial cells thus providing immunological protection plus nutritional and psychological benefits for the infant.

The Role of AIRE in the Immunity Against Candida Albicans in a Model of Human Macrophages.

Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a primary immunodeficiency caused by mutations in the autoimmune regulator gene (AIRE). Patients with AIRE mutations are susceptible to Candida albicans infection and present with autoimmune disorders. We previously demonstrated that cytoplasmic AIRE regulates the Syk-dependent Dectin-1 pathway. In this study, we further evaluated direct contact with fungal elements, synapse formation, and the response of macrophage-like THP-1 cells to C. albicans hyphae to determine the role of AIRE upon Dectin receptors function and signaling. We examined the fungal synapse (FS) formation in wild-type and AIRE-knockdown THP-1 cells differentiated to macrophages, as well as monocyte-derived macrophages from APECED patients. We evaluated Dectin-2 receptor signaling, phagocytosis, and cytokine secretion upon hyphal stimulation. AIRE co-localized with Dectin-2 and Syk at the FS upon hyphal stimulation of macrophage-like THP-1 cells. AIRE-knockdown macrophage-like THP-1 cells exhibited less Dectin-1 and Dectin-2 receptors accumulation, decreased signaling pathway activity at the FS, lower C. albicans phagocytosis, and less lysosome formation. Furthermore, IL-1ß, IL-6, or TNF-α secretion by AIRE-knockdown macrophage-like THP-1 cells and AIRE-deficient patient macrophages was decreased compared to control cells. Our results suggest that AIRE modulates the FS formation and hyphal recognition and help to orchestrate an effective immune response against C. albicans.

Protective activity of the antilipopolysaccharide antibodies from human cord serum.

We evaluated the ability of human maternal and cord serum antibodies to protect mice challenged with live Escherichia coli serotype O6:K2ac (E. coli O6). Mice received paired maternal or cord serum pools before a challenge with E. coli O6 to evaluate the mortality rate. All the pools were able to protect the animals challenged with bacteria except the test group from paired maternal and cord sera from preterm neonates containing less than 1.0 mg L(-1) immunoglobulin G antibody levels. In liver, spleen and mesenteric lymph nodes from the control group (phosphate-buffered saline), more than 10(2) CFU mL(-1) bacteria were found at 30 min and more than 10(5) CFU mL(-1) after 120 min. The test group showed lower bacterial counts in the organs, and no bacteria in the mesenteric lymph nodes during the evaluated period. Tumor necrosis factor alpha and interleukin 6 were undetectable in serum from animals pretreated with paired maternal and cord serum pools from full-term neonates and pools from preterm neonates containing high antibody and avidity levels. Our findings suggest that placental transfer of antilipopolysaccharide O6 immunoglobulin G antibodies to neonates has a high capacity to prevent lethal infection with E. coli O6 in a mouse protection model and that the degree of protection is determined by the concentration and avidity of these IgG antibodies.

Early Exposure to Respiratory Allergens by Placental Transfer and Breastfeeding.

The relationship between allergen exposure and the onset of or protection from allergic diseases remains unclear. Many factors could be related to immunological responses, such as the age when the exposure occurs, type of allergen, timing, dose, and allergen route. In this study, we investigated whether exposure to respiratory allergens could occur in pregnancy or early life. In particular, we assessed whether Der p 1 and Blo t 5, as well as specific antibodies against these allergens, could be detected in 90 paired cord blood and colostrum samples. Der p 1 was detected in 58.6% of colostrum and 29% of cord blood samples, whereas Blot 5 was positive in 41.3% and 9.6% of the samples, respectively. Similar to specific IgA, which could be detected in all samples for both mites, specific IgG was found in a high number of colostrum samples, 93.5% and 94.8% for Dp and Bt, respectively. Although allergens were not detected in all cord blood samples, a high percentage of them (≥95%) were positive for specific IgM to both mites in cord blood samples, suggesting that neonates can be exposed and sensitized to airborne allergens during pregnancy. Many studies have attempted to correlate allergen exposure or its prevention in early infancy with the onset of or protection from allergic diseases. However, conflicting and inconsistent data do not show a clear correlation with or suggest a way to prevent allergen sensitization. Nevertheless, these unconvincing results could be better understood if the relationship with many aspects of allergen exposure after pregnancy could be clarified. Thus, it is necessary to address basic issues related to allergen exposure, including the development of reproducible, standardized and reliable methods, and to determine how and where the exposure occurs.

The Role of M. leprae Hsp65 Protein and Peptides in the Pathogenesis of Uveitis.

Experimental autoimmune uveitis (EAU) is a well established model for immune-mediated organ-specific disease. Our group has recently shown that the M. leprae Hsp65 aggravated the uveitis in mice; in the present study, we evaluated the action of M. leprae K(409)A mutant protein and the synthetic peptides Leader pep and K(409)A pep (covering amino acids residues 352-371 of WT and K(409)A proteins of M. leprae Hsp65, resp.) on the pathogenesis of EAU. Mice received the 161-180 IRBP peptide and B. pertussis toxin followed by the intraperitoneal inoculation of K(409)A protein or the Leader pep or K(409)A pep. The Leader pep aggravated the disease, but mice receiving the K(409)A pep did not develop the disease and presented an increase in IL-10 levels by spleen cells and a decrease in the percentage of CD4+ IFN-γ+ T cells. Moreover, animals receiving the Leader pep presented the highest scores of the disease associated with increase percentage of CD4+ IFN-γ+ T cells. These results would contribute to understanding of the pathogenesis of EAU and support the concept that immune responses to Hsp are of potential importance in exacerbating, perpetuating, or even controlling organ-restricted autoimmune diseases, and it is discussed the irreversibility of autoimmune syndromes.

Minimal concentration of human IgM and IgG antibodies necessary to protect mice from challenges with live O6 Escherichia coli.

This work evaluated the ability of human anti-lipopolysaccharide O6 IgM and IgG antibodies to protect mice challenged with Escherichia coli serotype O6 : K2ac. Purified IgM-effluent, purified IgG, pools of normal human serum (NHS), or control group were injected into mice 18 h before challenges with O6 E. coli. Interleukin 6 and tumor necrosis factor alpha were quantified in the sera of test and control groups. All mice receiving purified IgM-effluent (66.6 mg L(-1) of anti-lipopolysaccharide O6 IgM antibodies) and NHS survived. Purified IgG (1.1 mg L(-1) of anti-lipopolysaccharide O6 IgG antibodies) protected 87.5% of the animals. The control group showed no protective ability. The minimal concentration of anti-lipopolysaccharide O6 IgM antibodies, able to protect 50% of the animals was 33.3 mg L(-1) of purified IgM-effluent, whereas purified IgG was able to protect 50% of the animals with only 1.1 mg L(-1) of anti-lipopolysaccharide O6 IgG antibodies. Serum from animals pretreated with purified IgM-effluent and purified IgG before challenges with lipopolysaccharide O6 did not have detectable pro-inflammatory cytokines. Hepatocytes of the control group were completely invaded by bacteria, whereas none was found in animals pretreated with purified IgM-effluent and purified IgG. Higher concentrations of anti-lipopolysaccharide O6 IgM antibodies as compared to anti-lipopolysaccharide O6 IgG antibodies were needed to protect mice from challenges with E. coli O6 serotype.

4-fluoro-2-methoxyphenol, an apocynin analog with enhanced inhibitory effect on leukocyte oxidant production and phagocytosis.

Apocynin, a methoxy-substituted catechol (4-hydroxy-3-methoxyacetophenone), originally extracted from the roots of Picrorhiza kurroa, has been extensively used as a non-toxic inhibitor of the multienzymatic complex NADPH oxidase. We discovered that the analogous methoxy-substituted catechol, 4-Fluoro-2-methoxyphenol (F-apocynin), in which the acetyl group present in apocynin was changed to a fluorine atom, was significantly more potent as an inhibitor of NADPH oxidase activity, myeloperoxidase (MPO) chlorinating activity and phagocytosis of microorganisms by neutrophils; it was also as potent as apocynin in inhibiting tumor necrosis factor-alpha (TNFα) release by peripheral blood mononuclear cells. We attribute the increased potency of F-apocynin to its increased lipophilicity, which could facilitate the passage of the drug through the cell membrane. The inhibition of MPO chlorination activity, phagocytosis and TNFα release shows that apocynin and F-apocynin actions are not restricted to reactive oxygen species inhibition, but further studies are needed to clarify if these mechanisms are related. Like apocynin, F-apocynin did not show cell toxicity, and is a strong candidate for use in the treatment of inflammatory diseases.

Evaluation of experimental autoimmune uveitis in mice treated with FTY720.

PURPOSE: FTY720 (fingolimod) is an immunomodulatory drug capable of preventing T-cell migration to inflammatory sites by binding to and subsequently downregulating the expression of sphingosine-1 phosphate receptor 1 (S1P(1)) leading in turn to T-cell retention in lymphoid organs. Additional effects of FTY720 by increasing functional activity of regulatory T cells have recently been demonstrated, raising the conversion of conventional T cells into regulatory T cells and affecting the sequestration of regulatory T cells in normal mice. In this study, the action of FTY720 in the ocular autoimmune model in mice was investigated. METHODS: Mice were immunized with 161-180 peptide and pertussis toxin and were treated with 1 mg/kg/d FTY720 by gavage (7-21 days postimmunization [dpi]) or left untreated. Spleen cells, harvested 21 dpi, were cultured and assayed for cytokine production. Draining lymph node, spleen, and eye cells 21 dpi were assayed for quantification of T-cell populations. Disease severity was evaluated by histologic examination of the enucleated eyes at 21 and 49 dpi. In addition, anti-IRBP antibodies were analyzed by ELISA. RESULTS: FTY720 was effective in suppressing the experimental autoimmune uveitis score. Although there was a reduction in the number of eye-infiltrating cells, FTY did not prevent Treg accumulation at this site. FTY720 leads to a significant increase of CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cell percentages in lymph nodes, suggesting that this site could be the source of Treg cells found in the eye. CONCLUSIONS: The data showed that treatment in vivo with FTY720 was able to suppress EAU in mice. These results are indicative of the possible therapeutic use of FTY720 in ocular autoimmune processes.

Evaluation of humoral response to heptavalent pneumococcal conjugate vaccine in HIV-infected children.

OBJECTIVE: Invasive pneumococcal disease is a major cause of death in HIV-infected children. The objective of the study was to assess the quantitative antibody response to the seven pneumococcal serotypes of heptavalent pneumococcal conjugate vaccine in a group of HIV-infected children. METHODS: Study comprising 40 HIV-infected children aged between 2 and 9 years followed up in a specialized outpatient clinic in São Paulo, Brazil, between 2002 and 2003. Enzyme immunoassay (ELISA) was used to measure IgG antibody titers against pneumococcus capsule. Antibodies were measured immediately before and 1 month after the second dose of the vaccine. Two response criteria were used: IgG titers >or=1.3 microg/mL in the post-immunization serology and an increase of at least 4or=-fold in post- compared to pre-immunization serology. RESULTS: For the first criterion (>or=1.3 microg/mL), 26 (65%) children had serological response to the vaccine, 12 (30%) showed post-immunization IgG titers of at least 1.3 microg/mL for all seven serotypes studied. For the second criterion studied (>or=4-fold increase in post- compared to pre-immunization titers for four serotypes or more), serological response was seen in 15 (37.5%) children. CONCLUSIONS: Overall response to the heptavalent pneumococcal conjugate vaccine was adequate, showing a statistically significant increase in the post-immunization geometric mean titers for the seven serotypes studied.

Passive immunity acquisition of maternal anti-enterohemorrhagic Escherichia coli (EHEC) O157:H7 IgG antibodies by the newborn.

Enterohaemorrhagic Escherichia coli (EHEC) strains are among the main causes of haemorrhagic colitis (HC) and haemolytic-uremic syndrome (HUS) in industrialised countries. In Brazil, EHEC have been detected in the faeces of patients with non-bloody diarrhoea, though an association between EHEC and HUS has been detected recently. These observations suggest that there is a pre-existing immunity triggered by the contact with EHEC and other categories of bacteria, such as EPEC, that share similar virulence factors and to which our population is highly exposed. Our aim was to evaluate the placental transfer of IgG antibodies reactive to EHEC O157:H7 antigens. We evaluated 28 paired maternal and cord sera for the presence of IgG against EHEC O157:H7 protein antigens and IgG and IgM to O157 LPS employing ELISA and IB technique. Total IgG and IgM level analyses were also made. Anti-EHEC O157:H7 and anti-LPS O157 IgG antibody levels in cord sera were equivalent to those of their maternal sera. A good correlation between the mothers' anti-LPS O157 IgM and total IgM levels was found. Anti-LPS O157 IgM levels were higher than anti-LPS O157 IgG levels in the same samples, and anti-LPS IgM antibodies were not detected in cord sera. Identical patterns of recognition of bacterial protein antigens by specific IgG were found in the paired samples and the recombinant purified variable region of gamma intimin was specifically recognized by one paired maternal and cord sample. In conclusion, although the antibody profile varied among individuals, all paired cord and maternal serum samples showed an identical recognition pattern, indicating an efficient placental transfer of IgG antibodies reactive to EHEC O157:H7 antigens.

Avaliação da resposta humoral à vacina pneumocócica 7-valente em crianças com Aids / Evaluation of humoral response to heptavalent pneumococcal conjugate vaccine in HIV-infected children / Evaluación de la vacuna neumocócica 7-valente en la respuesta humoral de niños con SIDA

OBJETIVO: A doença pneumocócica invasiva é importante causa de morbi-mortalidade em crianças infectadas pelo HIV. O objetivo do estudo foi avaliar quantitativamente a resposta com anticorpos aos sete sorotipos pneumocócicos da vacina em um grupo de crianças infectadas pelo HIV. MÉTODOS: Estudo realizado com 40 crianças infectadas pelo HIV, com idade entre 2 e 9 anos, em seguimento em ambulatório especializado no município de São Paulo, em 2002-2003. A dosagem de anticorpos IgG contra os polissacarídeos da cápsula pneumocócica foi realizada por meio de ensaio imunoenzimático (ELISA). Os anticorpos foram dosados imediatamente antes e um mês após a aplicação da segunda dose da vacina. Utilizaram-se dois critérios para avaliar a resposta à vacina: títulos de anticorpos >1,3 µg/mL na sorologia pós-imunização e aumento >4 vezes nos títulos da sorologia pós em relação à pré-imunização. RESULTADOS: Para o primeiro critério (>1,3 µg/mL), 26 (65 por cento) crianças obtiveram resposta sorológica à vacina, 12 (30 por cento) delas apresentaram títulos de IgG pós-imunização em níveis de pelo menos 1,3 µg/mL para todos os sorotipos. Para o segundo critério (incremento >4 vezes nos títulos para quatro sorotipos ou mais), obteve-se resposta sorológica para 15 (37,5 por cento) crianças. CONCLUSÕES: A resposta à vacina foi considerada satisfatória, com aumento estatisticamente significante dos títulos geométricos médios pós-vacinais em relação aos pré-vacinais para todos os sorotipos estudados.

Aquisição passiva de anticorpos anti-dermatophagoides pteronyssinus através de passagem transplacentária e aleitamento materno / Passive acquisition of anti-dermatophagoides pteronyssinus antibodies through placental transfer and breastfeeding

Introdução e objetivos: Eventos nos primeiros anos de vida podem ser responsáveis pelo aumento da prevalência de doenças alérgicas. Tendo em vista que o sistema imunológico da criança recebe anticorpos específicos a alérgenos ainda no útero e através da amamentação, caracterizamos a transferência passiva de IgG e IgA anti-Dermatophagoides pteronyssinus e verificamos o efeito da sensibilização materna na resposta imune humoral no cordão umbilical e no colostro. Métodos: Colostro e amostras pareadas de soro materno e de cordão umbilical foram coletadas de treze mães sensibilizadas (RAST anti-Der p > classe 3) e 25 mães não sensibilizadas (RAST anti-Der p = O). Quantificamos os níveis totais de IgG por nefelometria e a IgA total e os anticorpos específicos anti¬Der p por ELISA. Para análise funcional, verificamos a avidez dos anticorpos ao extrato total do ácaro, também por ELISA. Resultados: Recém-nascidos de mães sensibilizadas apre¬sentaram níveis significativamente mais elevados de IgG anti¬Derp no cordão umbilical (p=O,Ol), no entanto, estes anticor¬pos foram detectados em todas as amostras, estando fortemente correlacionados aos níveis maternos (r=0.81 p

Introduction and objectives: The early life sensitization might be one of the primary causes of the increased prevalen¬ce of allergy. Considering that infants received in the uteri and by the breastfeed specific antibodies to allergens, we investigated the passive transference of IgG and IgA antibodies to Dermatophagoides pteronyssinus to verify if mother sensitization influences the humoral immune response in umbilical cord blood and in colostrum...