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RT-PCR & real-time RT-PCR
applications in
Short-term
changes of mRNA expression of various inflammatory factors and milk
proteins in mammary tissue during LPS-induced mastitis
S. Schmitz, M.W. Pfaffl, H.H.D. Meyer, R.M. Bruckmaier (2004) Domestic Animal Endocrinology 26(4) 111-126  During mammary gland infection,
non-specific responses are the
predominant ones. The goal of this studywas to investigate
themRNAexpression of various soluble immune components and of the major
milk proteins during the acute phase of mammary inflammation. Five
healthy lactating cows were intramammary infused in one quarter with
100µg Escherichia coli-endotoxin (lipopolysac- charide, LPS) and
the contralateral quarter with saline (9 g/l) serving as control.
Mammary biopsy samples of both quarters were taken immediately before
and at 3, 6, 9 and 12 h after infusion and mRNA expression of various
factors was quantified via real-time RT–PCR. Blood samples for
determination of leukocyte number were taken simultaneously with the
biopsy samples
and rectal temperature was measured at 1-h intervals. Rectal
temperature
increased until 5 h (P < 0.05) after LPS administration and remained
elevated until 9 h after LPS inoculation. Blood leukocyte number
decreased
(P < 0.05) from 0 to 3 h from 7.7±1.1×109 l−1 to
5.7±1.0×109 l−1 and thereafter recovered to pre-treatment
levels until 12 h after LPS challenge. In LPS-treated quarters, tumor
necrosis factor-alpha and cyclooxygenase-2-mRNA expression increased (P
< 0.05) to highest values at 3 h after LPS
challenge. Lactoferrin, lysozyme, inducible nitric oxide synthase
increased
(P < 0.05) and peaked at 6 h after challenge, and
platelet-activating
factor acetylhydrolase-mRNA expression tended to increase (P = 0.07).
mRNA expression of insulin-like growth factor-I and of alphaS1-casein
(CN), alphaS2-CN, beta-CN and beta-lactoglobulin did not change
significantly,
whereas mRNA expression of 5-lipoxygenase and alpha-lactalbumin
decreased
(P < 0.05) in both quarters and that of kappa-CN only in the LPS
quarter.
mRNAexpression of some investigated factors (tumor necrosis
factor-alpha,
lysozyme, 5-lipoxygenase,beta-lactalbumin) changed in control quarters,
however in all respective factors less than in the LPS quarters (P <
0.05).
In conclusion, mRNA expression of most inflammatory factors increased
within
hours, whereas that of most milk proteins remained unchanged.
mRNA expression of immune factors and milk proteins in mammary tissue and milk cells and their concentration in milk during subclinical mastitis S. SCHMITZ, M.W. PFAFFL, M. MILLER, J. BUCHBERGER, T. MEYER, H. SAUERWEIN, R.M. BRUCKMAIER Milk Science International 59 (7/8) 2004 5-Lipoxygenase, Cyclo-oxygenase-2 and Tumor necrosis factor alpha (TNF-alpha) gene expression in somatic milk cells Wittmann SL,
Pfaffl M., Meyer HHD & Bruckmaier RM (2002) Summary The goal of this
work was to investigate whether different fractions of somatic cells in
cow milk do produce prostaglandins, leukotrienes and the cytokine tumor
necrosis factor alpha (TNFalpha). mRNA expressions of TNFalpha
and of key enzymes of prostaglandin and leukotriene biosynthesis,
cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), respectively, were
quantitatively
determined. Eleven clinically healthy Brown Swiss cows were either
defined
as control (C) group (n=5; all quarters < 150 000 cells/ml)
or as group with partially elevated quarter somatic cell counts (SCC;
n=6) with at least one quarter > 150 000 cells/ml (H) and one
quarter < 150 000 cells/ml (L). Total quarter milk from one quarter
of control animals and from two quarters of cows with partially
elevated
SCC (one of H and one of L) was collected. Cells were fractionized into
macrophages and lymphocytes (MAC+LYM) and polymorphonuclear leukocytes
(PMN) using a density gradient. RNA was isolated, reversely transcribed
and quantitative PCR was carried out. mRNA expression was similar for
5-LO,
COX-2 and TNFalpha: no differences between C and L quarters were
detected.
However, expression was markedly elevated in H quarters. mRNA
expression
of all parameters tested was higher in MAC+LYM than in the PMN
fraction.
In conclusion, this investigation underlines that somatic milk cells
are
able to synthesize 5-LO, COX-2 and TNFalpha.
in Blood Cells, Milk Cells and Mammary Tissue of Cows M. W. Pfaffl, S.
L. Wittmann, H. H. D. Meyer & R. M. Bruckmaier (2003)
Summary Cytokines,
eicosanoids and lactoferrin are involved in the mammary gland´s
immune response to invading microorganisms. The goal of this work
was to investigate the synthesis of these immunologically important
factors in somatic milk cells, blood cells and mammary tissue of cows
with different somatic cell count levels, i.e. different immunological
activity. On the level of mRNA expression, the cytokine tumor necrosis
factor a (TNFa), lactoferrin (Lf) and specific key enzymes of
leukotriene
and prostaglandin biosynthesis, 5-lipoxygenase (5-LO) and
cyclooxygenase-1
(COX-1) and –2 (COX-2), respectively, were determined. All fifteen
experimental
cows were clinically healthy with no visible mammary disease. Eight
cows
were defined as control group with all quarters < 150 000 cells/ml
(C)
while seven cows had partially elevated quarter somatic cell counts,
with
at least one quarter > 150 000 cells/ml (H) and one quarter
<
150 000 cells/ml (L). Total quarter milk from one quarter of control
group
and from two quarters of cows with partially elevated cell counts (one
of H and one of L) was collected at one milking and a blood sample was
taken simultaneously. In addition, mammary tissue samples were taken
from
the respective quarters on the following day during slaughter. Total
RNA
from milk, blood and tissue cells was isolated and reverse
transcription
and quantitative polymerase chain reaction (RT-PCR) was carried out.
All
factors investigated were not significantly different between groups in
blood cells and between C and L quarters in milk cells and mammary
tissue. TNFa and COX-2 mRNA expression was higher in milk cells and
mammary tissue of H than in L quarters, except for COX-2 in mammary
tissue. Generally TNFa and COX-2 showed their highest expression in
milk cells, 5-LO in blood cells, whereas lactoferrin was mainly
expressed by the mammary tissue. COX-1
was similarly expressed in all tested samples.
Acute
Phase Inflamatory Reaction In The Bovine Mammary Gland Mammary
Gland Health and Disease Multidisciplinary
joint meeting |
