Aflatoxin B1 and M1 contamination of animal feeds and milk from urban centers in Kenya.

BACKGROUND: Aflatoxin M1 (AFM1) is the principal hydroxylated AFB1 metabolite present in milk of cows fed with a diet contaminated with AFB1and excreted within 12 hours of administration of contaminated feeds. OBJECTIVE: This study was initiated to assess the knowledge and practices of urban dairy farmers and feed millers about aflatoxin in feeds and milk, determine the prevalence and quantify the levels of AFB1 and AFM1 in animal feeds and milk respectively from urban environs in Kenya. METHODS: This work was carried out in the Department of Public Health Pharmacology and Toxicology, University of Nairobi, Kenya, between February 2006 and March 2007. RESULTS: A total of 830 animal feed and 613 milk samples from four urban centers were analyzed for aflatoxin B1 and M1 respectively using competitive enzyme immunoassay. Eighty six percent (353/412) of the feed samples from farmers were positive for aflatoxin B1 and 67% (235/353) of these exceeded the FAO/WHO level of 5µ gKg-1. Eighty one percent (197/243) of the feed samples from feed millers and 87% (153/175) from agrochemical shops were positive, while 58% (115/197) and 66% (92/153) of the positive samples exceeded the FAO/WHO limits respectively. Seventy two percent (315/439) of the milk from dairy farmers, 84% (71/85) from large and medium scale farmers and 99% (88/89) of the pasteurized marketed milk were positive for aflatoxin M1, and 20%, 35% an 31% of positive milk from dairy farmers, medium and large scale farmers and market outlets respectively, exceeded the WHO/FAO levels of 0.05µ g/Kg-1. Sixty seven percent of the urban smallholder dairy farmers had no knowledge that milk could be contaminated with aflatoxin M1 and neither knew how they could mitigate against this exposure. Feed millers knew about aflatoxin B1 in grains and excretion of aflatoxin M1 in milk, but were not alleviating exposure to animals. CONCLUSION: There is need to create awareness and establish routine monitoring of animal feeds and milk to reduce animal and consequently human response.

Abnormal TGFbeta levels in the amniotic fluid of Down syndrome pregnancies.

PROBLEM: The status of cytokines in amniotic fluid (AF) from chromosomally abnormal pregnancy is largely undefined. TGFbeta plays a key role in fetal growth and differentiation and is responsible for the immunoregulatory activity of AF in normal pregnancy, but its status in Down syndrome (DS) pregnancies is unknown. In addition we investigated the IL-2 status of AF from DS pregnancies. METHOD OF STUDY: Midtrimester AF from chromosomally normal (n = 25) and abnormal pregnancies with DS (n = 15) were assayed for bioactive and latent TGFbeta levels using the mink lung epithelial cell growth inhibition bioassay and for IL-2 activity by the CTLL-2 cell proliferation bioassay and by ELISA. RESULTS: Levels of bioactive TGFbeta (mean 4.6+/-0.6 U/mL) were significantly increased in DS AF compared with the normal samples (mean 2.8+/-0.3 U/mL, P<0.003) but latent TGFbeta levels did not differ between DS and normal groups. In addition DS AFs showed reduced IL-2 like bioactivity compared with normal samples (P = 0.0006) but IL-2 immunoactivity was undetectable in DS and normal AFs by ELISA. CONCLUSIONS: DS AFs display increased TGFbeta activity and lacked IL-2 immunoactivity. The reduced ability of DS AFs to stimulate CTLL-2 cell proliferation is unrelated to the IL-2 status of AF. Altered TGFbeta levels may prove useful as an additional biochemical index for the detection of DS pregnancies.

Human amniotic fluid lacks interleukin-2 and interleukin-15 but can interact with the beta-chain of the interleukin-2 receptor.

The present study investigated whether an explanation for the conflicting reports on the interleukin-2 (IL-2) status of amniotic fluid is due to the presence of IL-15 which shares biological activities with IL-2 and utilizes the IL-2 receptor beta-chain. Amniotic fluids from 45 normally progressing pregnancies between 14 and 16 weeks after the last menstrual period were assayed for IL-2 and IL-15 by bioassay and enzyme-linked immunosorbent assay (ELISA). The ability of amniotic fluids to induce cytotoxic T lymphoblastoid line-2 (CTLL-2) cell proliferation was demonstrated to be dependent upon bioassay culture conditions. In serum-free medium each amniotic fluid stimulated CTLL-2 proliferation with a mean level of IL-2-like bioactivity of 14.7 +/- 2.3 ng/ml but amniotic fluids failed to induce CTLL-2 proliferation in serum-supplemented medium. Treatment with neutralizing anti-IL-2 or anti-IL-15 antibodies failed to inhibit amniotic fluid-induced CTLL cell proliferation in serum-free medium, indicating a lack of IL-2 and IL-15 bioactivity. In contrast, treatment with anti-IL-2 receptor beta-chain antibody significantly reduced amniotic fluid-induced proliferation. The lack of IL-2 and IL-15 activity in amniotic fluids was confirmed using ELISA. Although high levels of IL-15 immunoactivity were detected in all samples, specificity controls showed a lack of specific IL-15 immunoactivity in amniotic fluid. Pretreatment of amniotic fluids with 100-500 ng/ml mouse immunoglobulin G abrogated IL-15 immunoactivity, indicating that amniotic fluid contains molecules binding to Fc regions of immunoglobulins and responsible for false ELISA positivity. These studies unequivocally show that amniotic fluid lacks IL-2 and IL-15 but can stimulate CTLL-2 cell proliferation via the IL-2 receptor beta-chain. The absence of IL-2 and IL-15 in normal mid-trimester amniotic fluid suggests that the cytokine profile of human pregnancy appears to be associated with a bias against type 1 cytokines within the feto-placental unit.

Ability of T cells from patients with rheumatoid arthritis to respond to immunoglobulin G.

The ability of T cells from rheumatoid factor (RF)-positive patients with rheumatoid arthritis (RA) to respond to immunoglobulin G (IgG) was assessed. Peripheral blood mononuclear cells (PBMC) from RA patients and normal individuals were cultured with and without human IgG or Mycobacterium tuberculosis-purified protein derivative (PPD) for 7 days and their proliferative response measured at intervals by their ability to take up tritiated thymidine. PBMC from 14/26 RA patients proliferated in response to IgG (taking a stimulation index of 3 or above as positive). The peak response varied between individuals but usually occurred on day 5, the same day, or 1 day later than the peak response to PPD. By contrast, PBMC from a significantly lower proportion (1/9) of normal individuals and patients with other arthritides (0/6) responded to IgG, although all responded to PPD. PBMC from 9/14 RA patients responded to Fab fragments of IgG but only 3/9 to the Fc fragment. Higher proliferative responses from RA PBMC were elicited by IgG aggregates than the original IgG preparation, but PMBC from 5/5 normal individuals and 5/6 patients with other arthritides failed to respond to the aggregates. The response to IgG was human leucocyte antigen (HLA)-DR restricted and mediated by CD4+ T cells. It is considered that these results advance the hypothesis that IgG-reactive T cells contribute to the initiation or perpetuation of RA.

Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin.

We demonstrate that the receptor binding moiety of Escherichia coli heat-labile enterotoxin (EtxB) can completely prevent autoimmune disease in a murine model of arthritis. Injection of male DBA/1 mice at the base of the tail with type II collagen in the presence of complete Freund's adjuvant normally leads to arthritis, as evidenced by inflammatory infiltration and swelling of the joints. A separate injection of EtxB at the same time as collagen challenge prevented leukocyte infiltration, synovial hyperplasia, and degeneration of the articular cartilage and reduced clinical symptoms of disease by 82%. The principle biological property of EtxB is its ability to bind to the ubiquitous cell surface receptor GM1 ganglioside, and to other galactose-containing glycolipids and galactoproteins. The importance of receptor interaction in mediating protection from arthritis was demonstrated by the failure of a non-receptor-binding mutant of EtxB to elicit any protective effect. Analysis of T cell responses to collagen, in cultures of draining lymph node cells, revealed that protection was associated with a marked increase in interleukin 4 production concomitant with a reduction in interferon gamma levels. Furthermore, in protected mice there was a significant reduction in anti-collagen antibody levels as well as an increase in the IgG1/IgG2a ratio. These observations show that protection is associated with a shift in the Th1/Th2 balance as well as a general reduction in the extent of the anti-type II collagen immune response. This suggests that EtxB-receptor-mediated modulation of lymphocyte responses provides a means of preventing autoimmune disease.

The immunomodulatory activity of human amniotic fluid can be correlated with transforming growth factor-beta 1 (TGF-beta 1) and beta 2 activity.

The role of alphafetoprotein (AFP) in the immunomodulatory activity of amniotic fluids (AF) from normally progressing human pregnancy (weeks 14-16) was investigated. A panel of 42 AF (25% v/v) reduced significantly phytohaemagglutinin (PHA)-induced peripheral blood mononuclear cell (PBMC) proliferation in serum-free cultures with a mean per cent inhibition of 68.4 +/- 5.5%. In contrast, AFP preparations, with one exception (U.AFP), failed to display inhibitory activity. Pretreatment of AF with anti-TGF-beta 1 and beta 2 antibodies used alone resulted in the mean per cent loss of inhibition of 33.1 +/- 3.9% and 52.3 +/- 7.5%, respectively. A summative loss of AF-mediated inhibition was detected when anti-TGF-beta 1 and beta 2 antibodies were used in combination, but immunomodulation was rarely abolished 100% by this treatment. Anti-TGF-beta 2 antibody treatment, unlike anti-TGF-beta 1 antibody treatment, reversed the inhibitory activity of U.AFP. The amount of TGF-beta 1 and beta 2 contained in human AF was studied by growth inhibition of Mv1 Lu cells. The mean levels of TGF-beta 1 and beta 2 in AF were 11 +/- 0.9 U/ml and 2.3 +/- 0.4 U/ml, respectively, which corresponds with a mean per cent inhibition of 49 +/- 4.7%. U.AFP also significantly inhibited Mv1 Lu cell growth. To investigate the mechanism of AF-mediated inhibition, the effect of AF and AFP on IL-2 production by concanavalin A (Con A)-stimulated PBMC blasts was determined by the CTLL-2 cell bioassay. IL-2 production was reduced 55.5% in AF-treated blasts and 61% in U.AFP-treated blasts compared with controls. Our findings indicate that the immunomodulatory activity of human AF can be correlated with TGF-beta 1 and beta 2 and not with AFP, the inhibitory activity of U.AFP preparation reflecting copurifying TGF-beta 2 activity.