galectin-3 is a -galactoside - binding animal lectin that contains carbohydrate - recognition domains and displays multiple related functions .
extracellular galectin-3 mediates inflammation , whereas intracellular galectin-3 regulates cell growth and anti - apoptosis , and modulates cell adhesion , thus inducing cell migration .
galectin-3 has been found in the cytoplasm of various cell types , including inflammatory cells such as macrophages , dendritic cells , mast cells , neutrophils , and eosinophils .
the expression of galectin-3 is known to be upregulated following certain bacterial   and parasitic infections .
in addition , it is accumulated in phagosomes containing mycobacterium during the course of an infection . here , we propose that galectin-3 is associated with the formation of granulomatous inflammatory lesions in chronic diseases including paratuberculosis .
paratuberculosis , or johne 's disease , is a chronic , granulomatous enteritis found in wild and domestic ruminants .
paratuberculosis , which is a slow - growing facultative intracellular bacterium that persists within macrophages in the intestinal tract for several years before clinical onset .
the symptoms of clinical paratuberculosis are chronic diarrhea and progressive weight loss ; subclinically infected animals may display decreased production .
the gross lesions are characterized by a segmental thickening of the intestine and mesenteric lymphadenopathy .
the aim of this study was to examine the immunohistochemical localization of galectin-3 in granulomatous intestines of bovines infected with paratuberculosis to determine whether galectin-3 was involved in the formation of granulation tissue in chronic disease .
rpob , encoding the  subunit of rna polymerase , pcr - plasmid ta cloning - sequencing for mycobacterium species was used to diagnose the presence of paratuberculosis .
tissue samples ( n = 3 ) from the small intestines , mainly ileum , and mesenteric lymph nodes were obtained from cows at slaughter that were suspected to be infected with paratuberculosis .
tissue samples were fixed with 4% paraformaldehyde in phosphate - buffered saline ( pbs ) and processed for paraffin - embedding .
sections ( 5 m thick ) were cut in a microtome ( leica , germany ) , and were routinely examined with hematoxylin and eosin staining and with ziehl - neelsen staining for acid - fast bacilli . as a control , bovine ileum without acid - fast bacilli staining was used . for immunohistochemistry
, 5-m - thick sections of paraffin - embedded bovine ileum and mesenteric lymph nodes were deparaffinized using xylene and pure ethanol before exposed to citrate buffer ( 0.01 m , ph 6.0 ) .
the sections were treated with 0.3% hydrogen peroxide in methyl alcohol for 20 min to block endogenous peroxidase activity .
after three washes in pbs , the sections were blocked with 10% normal goat serum ( vector abc elite kit ; vector laboratories , usa ) , diluted in pbs for 1 h , and then incubated with rat anti - galectin-3 antibody ( 1 : 5,000 ) for 1 h. the rat anti - galectin-3 monoclonal antibody ( 1 mg / ml ) was purified from the supernatant of hybridoma cells ( tib-166 ; atcc , usa ) . after three washes in pbs , the sections were incubated with biotinylated goat anti - rat igg ( 1 : 100 ; vector laboratories , usa ) for 45 min . after three washes in pbs
, the sections were incubated with the avidin - biotin peroxidase complex ( vector laboratories , usa ) , prepared according to the manufacturer 's instructions , for 45 min .
the peroxidase reaction was developed using a peroxidase substrate kit ( vector laboratories , usa ) according to the manufacturer 's protocol .
after the completion of color development , the sections were counterstained with hematoxylin ( sigma , usa ) for 5 sec , washed in running tap water for 20 min , dehydrated through a graded ethanol series , and then cleared with xylene and mounted with canada balsam ( sigma , usa ) .
as a negative control , the primary antibody was omitted . for the diagnosis of paratuberculosis from paraffin embedded tissues , the paraffin - embedded specimens were deparaffinized with xylene and pure ethanol .
the pcr products obtained were electrophoresed in a 1.5% agarose gel and purified using a qiaex ii gel extraction kit ( qiagen , germany ) . the purified pcr product ( 5 - 10 ng )
was cloned using a ta cloning kit ( invitrogen , usa ) according to the manufacturer 's instructions .
three to 10 colonies of transformed escherichia coli were picked in each reaction , cultured , and used to prepare plasmid dna with a high pure plasmid isolation kit ( roche , germany ) .
the nucleotide sequences of the cloned rpob dnas were directly determined from the purified plasmid using m13 forward and reverse primers , which were supplied in the ta cloning kit , a 373a automatic sequencer , and a bigdye terminator cycle sequencing kit ( pe applied biosystems , uk ) . for the sequencing reaction ,
60 ng of pcr - amplified dna , 3.2 pmol of either the forward or the reverse primer , and 4 l of bigdye terminator rr mix ( pe applied biosystems , uk ) were mixed and adjusted to a final volume of 20 l by adding distilled water .
the reaction was run with 5% ( vol / vol ) dimethyl sulfoxide for 30 cycles of 15 sec at 95 , 10 sec at 50 , and 4 min at 60. both strands were sequenced as a cross - check .
the sequences determined ( 306 bp ) were aligned and compared to sequences in genbank by using the multiple - alignment algorithms in the megalign package ( windows version 3.12e ; dnastar , usa ) .
all three cases showed typical granulation tissues in the intestines with a varying degree of inflammation .
briefly , the lamina propria and submucosa of the small intestine was thickened due to the infiltration of inflammatory cells ( fig .
the thickening of the mucosa was attributable to the accumulation of typical lymphocytes with condensed nuclei and macrophages with foamy , pale cytoplasm ( fig .
an accumulation of multinucleated giant cells were also found in the mesenteric lymph nodes ( fig .
1c ) . in the acid - fast stained tissue sections of the small intestine ,
the presence of acid - fast bacilli was confirmed due to its red coloration ( fig .
acid - fast bacilli were found in various cell types , including macrophages , in the lamina propria and submucosa of the small intestine .
the lamina propria just below the epithelium of the villi was intensely stained red , suggesting that acid - fast bacilli were compacted in macrophages within this area ( fig .
2c ) . in the adjacent tissues of the acid - fast stained section , galectin-3
was strongly expressed in the round cells ( typical of macrophages ) in the lamina propria and submucosa of the intestines ( fig .
the rpob dna ( 368 bp ) was amplified from the small intestine sample , including ileum . however , due to weak amplification , pcr - direct sequencing was not possible .
therefore , the pcr products obtained were cloned into a ta plasmid for sequencing , and the inserted dna sequences of the sample were compared with the sequences of 44 reference strains of mycobacteria and sequences in the genbank database .
this study confirms for the first time the accumulation of galectin-3-positive macrophages in bovine paratuberculosis - infected granulation tissues , particularly the ileum .
galectin-3 has long been known as an important mediator of macrophage activation   and postulated to play a critical role in phagocytosis by macrophages .
however , the precise mechanisms of how galectin-3 interrupts bacilli digestion by phagosomes within macrophages during paratuberculosis infection remains unknown and requires further study .
in addition , galectin-3 has been known to be a major adhesion molecule   and involved in the trafficking of inflammatory leukocytes , including activated macrophages .
based on these previous studies , we hypothesize that macrophages continuously accumulate in the granulation tissues . following the accumulation of macrophages in the granulation tissues of paratuberculosis , macrophages were found to be alive in the lesions even though they contained bacilli .
bacilli - containing macrophages are thought to survive due to the anti - apoptotic characteristics of galectin-3 .
the molecular mechanism underlying this effect is cytochrome c release from the mitochondria , which possibly blocks cell death .
in contrast to the increase of galectin-3 in macrophages during paratuberculosis , galectin-3 decreases in the intestinal epithelia of animals with intestinal bowel disease , suggesting that the expression of galectin-3 is cell type - dependent .
in addition to the immunological role of galectin-3 in inflammatory cells , galectin-3 has been found in the epithelia of the digestive and respiratory tracts   as well as the reproductive organs , where mucus is abundant . in the present study
since galectin-3 is one of the ligands for mucin , we postulate that galectin-3 is involved in the mucin - mediated protective mechanisms of epithelial cells . in conclusion , we postulate that intracellular galectin-3 is involved in the protection of macrophages that contain bacilli , while extracellular galectin-3 is involved in the facilitation of inflammatory cell accumulation .
 the presence of galectin-3 was immunohistochemically quantified in bovine intestines infected with paratuberculosis ( johne 's disease ) to determine whether galectin-3 was involved in the formation of granulation tissue associated with the disease . 
 mycobacterium avium subsp . 
 paratuberculosis infection was histochemically confirmed using ziehl - neelsen staining and molecularly diagnosed through rpob dna sequencing . 
 galectin-3 was detected in the majority of inflammatory cells , possibly macrophages , in the granulomatous lesions within affected tissues , including the ileum . 
 these findings suggest that galectin-3 is associated with the formation of chronic granulation tissues in bovine paratuberculosis , probably through cell adhesion and anti - apoptosis mechanisms . 
Introduction
Materials and Methods
Results
Discussion