PNA-and SBA-positive lymphocytes content in the major salivary glands ’ structures during early postnatal period after intrauterine antigenic action

Background Salivary glands’ diseases have a varied etiology that includes autoimmune, infectious, idiopathic, and hereditary causes. The unifying factor in these diseases is the formation of granulomas, which histologically are mononuclear infl ammatory cells or macrophages surrounded by lymphocytes [5]. It is also known that salivary glands NK cells, although related to conventional NK cells, are a distinct subset of innate lymphoid cells that deviates from the conventional developmental pathway, perhaps under the infl uence of tissue-specific factors [4]. Improved the lymphocytes population infl uence on a formation of different organs and systems. Immature lymphocytes migrated from thymus to the peripheral organs of immune system [9]. One of the determinatives that result in violation of major salivary glands morphogenesis and as a result the development of its pathology is the condition of pregnant women health, more than half of them have chronic diseases and system functional disorders which are accompanied by the immune pathological condition, namely by antigen infl uence on a fetus [3,9]. In the literature there are no systematic data concerning the intrauterine antigen infl uence on morphogenesis of major UDC: 611.316.018.53:616-097.1]-053.31-092.9


Objective
To determine the PNA+ and SBA+ lymphocytes content in the major salivary glands' structures in early postnatal period after intrauterine antigen action.

Methods
The objects of the research were 224 salivary glands of white laboratory rats.Due to impossible qualitative materials taking in the early periods of postnatal life from parotid and sublingual salivary glands the investigation was carried out on the submandibular salivary glands.The rats were divided into three groups.First group are intact rats.Second group are rats, to which 0,05 ml solution of antigen were injected into the amniotic fl uid on the 18 th day of pregnancy by the method of N. Voloshyn [10], the third group -control, 0,05 ml of physiological solution were injected to the animals intrauterinely on the 18 th day of pregnancy.The feeding of animals was twice a day at the same time.For the study of peculiarities of PNA+ and SBA+ lymphocytes content in the major salivary glands structures after antigen's action on the fetus, the model of transuterine, transmembrane injection of antigen into amniotic fl uid by the method of N. Voloshyn was chosen [10].The antigen was liquid (killed) split -vaccine Vaxigrip 2009.Keeping the animals and experiments were carried out accordingly to regulations of European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientifi c Purposes (Strasbourg, 18.03.86),law of Ukraine «About protection of animals from cruel treatment» (№ 1759 from 15.09.2009).The animals killing and taking of the material were done from 13-00 till 14-00 on the 1 st , 5 th , 7 th , 11 th , 14 th , 30 th , 45 th days of postnatal life.On every term in all groups of the animals were examined 5-6 animals from 2-3 afterbirths.For the investigation the major salivary glands were used during some minutes after killing.The samples were fi xed in 10% solution of formalin, dehydrated, fi lled in paraffi n mixture and 4 μm thick serial paraffi n sections were produced.The sugar residues determination of β-D-galactose and N-acetyl-D-galactosamine was investigated using the standard method [2] through peanut agglutinin (PNA) and soybean agglutinin (SBA) using the standard kit of NPA «Lectin Test», Lviv.For visualization 3.3'-diaminobenzidine was used as the staining agent.The results of account of lectinhistochemistry were done semi-quantitatively and determined as +++ -dark brown stain, ++ -brown stain, +pale brown stain, zero is absence of stain.Intermediate tones were evaluated accordingly: ++/+++, +/++.PNA+ receptors distribution was detected on the intraepithelial lymphocyte plasmatic membrane and stromal lymphocytes of major salivary glands.SBA+ receptors were detected on the stromal lymphocyte plasmatic membrane [5].The PNA+ and SBA+ lymphocytes quantities were counted per notional area unit through A. Glagolev's modifi ed ocular grid with the fi ndings recalculation per 10000 μm 2 (1000 magnifi cation).Process-ing of the obtained numerical results was conducted through statistical methods using STATISTICA ® for Windows 6.1 (StatSoft Inc., № AXXR712D833214FAN5).Comparison of variables was performed using Student's t-test.
To verify existence of relationship between obtained variables correlation analysis (Pearson correlation coeffi cient) was used.The difference between the variables was considered statistically signifi cant at the p ≤ 0,05.
The work is the fragment of SRW of the Department of Anatomy, Operative Surgery and Topographic Anatomy and the Department of Histology, Cytology and Embryology of Zaporizhzhya State Medical University «Lectinhistochemical characteristics of morphogenesis of the organs and tissues in early postnatal period іn norm and experiment» (2008-2013, № state Registration 0109 U 003986).

Results
In the acini, PNA+ lymphocytes are situated among the serous and mucous secretory cells, often near the cells with mitosis fi gures.Intraepithelial lymphocytes are present mainly as the small forms.There are the round cells with a peripherally pigment deposition and pericytoplasmic enlightenment.
On the fi rst day after birth the PNA+ lymphocytes quantity in the intact group is 19,8±1,13.In antigenpremium rats the intraepithelial PNA+ lymphocytes content is increased compared with intact group -24,3±1,32.Indicators of all above-stated structures for the animals of control group do not differ from the data obtained from the animals of intact group, that is why in the future control group will not be cited (fi g. 1).
On the 5 th day of postnatal life, the quantity of intraepithelial lymphocytes with PNA receptors decreases in intact animals group compared with newborn rats.In experimental group the PNA+ lymphocytes content is signifi cantly more than in intact group -22,3±1,61 (p ≤0,05).On the 7 th day, in intact animals group PNA+ lymphocytes quantity decrease relative to the previous observation term is observed.In experimental group the tendency of PNA + lymphocytes content increase is saved compared with intact animals.On the 11 th day of postnatal life in observed animals from 1 st group the quantity of lymphocytes with β-D-galactose receptors is continuing to decrease gradually compared with previously observed term.In experimental group the PNA + lymphocytes content is remaining greater relative to the intact group (fi g. 1).On the 14 th day after birth in intact animals group the intraepithelial PNA+ lymphocytes quantity remains at the level of previously observed term but in experimental group the PNA+ lymphocytes content is statistically signifi cantly more compared with control animals.
The intraepithelial lymphocytes quantity on the 30 th day of postnatal life in intact group is somewhat reduced compared with the data from animals of 14 th day after birth.In experimental animals group the content of lymphocytes with PNA+ receptors is decreasing to the indicators in intact group of the current observation period.At the 45 th day of postnatal life, the PNA+ lymphocytes quantity remains at the level of previously observed term.Statistically significant is no difference between the indicators of investigated parameters (fi g. 1).
PNA -and SBA-positive lymphocytes content in the major salivary glands' structures during early postnatal period after ...

І. N. Maslova
PNA+ lymphocytes are situated among the fi broblasts of major salivary glands' stromal structures.On the 1 st day after birth in intact animals group the quantity of lymphocytes with PNA receptors is total 2,2±0,09 per 5000 μm 2 of the stromal structures.In experimental animals the PNA+ lymphocytes content was signifi cantly more than in intact group and was 3,1±0,22 per 5000 μm 2 .The intact group indicators signifi cantly do not differ from control group results (fi g. 2).
On the 5 th day of postnatal life in the control group the PNA+ lymphocytes quantities in the stromal structures increase relative to previously observed term.
In experimental group the quantity of the small lymphocytes with β-D-galactose residues remains statistically signifi cantly more (3,8±0,11) as compared with indicators of intact group (2,8±0,13).On the 7 th day of postnatal formation the quantity of lymphocytes with PNA receptors in all observed groups is the maximum during experiment.In antigenpremium animals their content remains more (4,1±0,1) relative to the control group (3,4±0,1; p≤ 0,05).On the 11 th day in intact group among the stromal cells the PNA+ lymphocytes quantity decrease is observed and their content is 2,3±0,1 per notional area unit.In the experimental group their quantity remains more than the results of control group and is 2,6±0,08 per notional area unit.At the 14 th day of postnatal life in the stromal structures of intact animals the PNA+ lymphocytes quantities decrease compared with previously observed term.In the experimental group their part remains more than indicators of control group 2,0±0,09.The PNA+ lymphocytes quantities decrease tendency is observed till the end of the experiment (fi g. 2).On the 30 th day the PNA+ lymphocytes content in experimental group is decreased to 1,4±0,1.The results obtained in the experimental group do not signifi cantly differ from the indicators in the control group.On the 45 th day of postnatal life a tendency to decrease of quantities of lymphocytes with PNA receptors in the rats' salivary glands' stromal structures remains in all investigated groups.The data statistically does not differ in all observed groups (fi g.2).
The SBA+ lymphocytes are located diffusely in the capsule, septa and ducts of the major salivary glands.In newborns animals of intact group the quantity of SBA+ lymphocytes with phenotypic features of small forms and intermediate forms is 1,9±0,1 per notional area unit.In experimental animals of the 1 st day after birth the lymphocytes with SBA+ receptors quantity decrease is observed compared with intact animals group.In the control group the SBA+ Fig. 1.PNA+ lymphocytes content in major salivary glands' parenchymal structures per notional area unit (10000 μm 2 ).Notes: ★ -result is signifi cant compared with intact group; # -result is signifi cant compared with previously observed term.
Fig. 2. PNA+ lymphocytes content in major salivary glands' stromal structures per notional area unit (10000 μm 2 ) according to the age and observation group.
Notes: ★ -result is signifi cant compared with intact group; ♯ -result is signifi cant compared with previously observed term.
lymphocytes quantity does not differ signifi cantly from the indicators of intact group from the neonatal period till the end of experiment.On the 5 th day in the intact group the tendency to increase of number of SBA+ lymphocytes is detected relative to the newborn animals (fi g. 3).In the experimental group the quantity of lymphocytes with N-acetyl-D-galactosamine residues on the receptors is 1,8±0,09 that is more than in newborn rats and signifi cantly less relative to the intact group.On the 7 th day the tendency to quantity increase of SBA+ cells with phenotypic features of small and intermediate lymphocytes forms in intact group remains relative to previously observed term.For experimental animals the indicators of SBA+ lymphocytes quantity remain lower as compared with the intact animals (fi g. 3).The 11 th day of postnatal life is characterized by increased quantity of lymphocytes with SBA receptors in intact group.The SBA+ lymphocytes content in the major salivary glands' connective tissue relative to the cells number is 2,7±0,1.For experimental animals group the total amount of SBA+ lymphocytes is lower than in the control group.On the 14 th day, in the major salivary glands' connective tissue of intact animals group the quantity of SBA+ cells with phenotypic features of the small and intermediate lymphocytes is increased as compared with previously observed term (fi g. 3).At fi rst, unlike the previously observed period, in experimental group the SBA+ lymphocytes quantity is 3,5±0,1, that is signifi cantly more than the results of intact animals.On the 30 th day after birth, the SBA+ lymphocytes quantity, as compared with indicators of the 14 th day, increases by 0,38 per notional area unit.Indicators of the lymphocytes with SBA+ receptors obtained from experimental group are at the level of the control group indicators (fi g. 3).On the 45 th day of postnatal formation, the quantity of SBA+ cells with phenotypic features of the small and intermediate lymphocytes in the major salivary glands' connective tissue is signifi cantly decreased relative to the previous observation term.The results obtained in experimental and intact groups during this observation period do not differ.

Discussion
Salivary gland mononuclear cells are composed of a variety of subpopulations distributions of which differ between parotid (PG) and submandibular salivary glands (SMG) and are distinct from lymphatic node, both PG and SMG were enriched in immature and activated T cells.Unchanged percentages of Thy-1(+) T cells in PG and SMG following short-term adult thymectomy indicated that immature salivary gland T cells had an extrathymic source [6].The SMG considered a privileged site where dissemination of the virus to other tissues as well as transmission to native individuals is possible.The SMG provides a peculiar barrier where mucosal tissues are typically poised to respond in a Th2 fashion and stimulate the production of IgA, while denying access to commensal microbes and other environmental pathogens [8].Sugar residues are important components of salivary gland secretion.Acinar cells contained abundant quantities of glycoconjugates with the terminal trisaccharide sialic acid -(alpha 2-3, 6) galactosyl (beta 1-3) N-acetylgalactosamine.Mandibular acinar cells also contained alpha and beta N-acetylgalactosamine and N-acetylglucosamine residues, whereas the demilunar cells contained glycoconjugates with fucose, mannose and Nacetylglucosamine residues.In the duct system a range of sugar residues was localized throughout the cell cytoplasm or limited to the apical surface [7].Secretions in parotid and submandibular serous cells generally contained a higher content of fucose than those in sublingual serous cells, which contained higher levels of a terminal galactose-sialic acid dimer.Some but not other cells of striated and interlobular ducts of submandibular glands of one subject were stained for alpha-N-acetylgalactosamine [1].
Our study showed that in group of animals after antigen action in fetal period the PNA+ lymphocytes quantity increase is observed.Among them there are two populations -immature forms and γ/δ lymphocytes [9].Thus, the PNA+ lymphocytes quantities dynamics have an undulatory character.In newborn animals group after intrauterine antigen action till the 7 th day of postnatal life the PNA+ lymphocytes number PNA -and SBA-positive lymphocytes content in the major salivary glands' structures during early postnatal period after ...