Chapter 9

                                                Chapter  9. 

Pathology of the Immune System in Human Fetuses and Newborns      Affected by Infectious Diseases

Infectious and inflammatory diseases are the most wide­spread reasons for pregnancy loss, stillbirth and neonatal death. Susceptibility of low birth weight (LBW) new­borns to infections derives from deficiencies in their cellular and humoral immunological mechanisms.  Studies of morphological and morphometric features of the immune reaction of fetuses and newborns have revealed fetal immune incompetence as one of the major reasons for their death or retarded postnatal develop­ment (1,2).

To better understand the role of the fetal immune system in the reaction of fetuses and newborns to infectious diseases, we studied the changes in the number of transcrip­tionally active nucleolar organizer regions (NORs) in splenic lymphoid cells, lymphocytes (resting cells) and lym­phoblasts (activated cells) of LBW and full-term fetuses and newborns with microbial infections such as broncho­pneumonia and sepsis (3). 

9.1)  Nucleolar organizer regions in the lymphoid cells of fetuses and newborns with bronchopneumonia and sepsis

NORs are the defined sites in the cell nucleus for the RNA synthesis on chromosomal matrices of DNA (4),  and NOR-­associated proteins reflect cell-cycle activity during cell dif­ferentiation and proliferation (5). These proteins are demonstrated in interphase nuclei by the argyrophilic (silver-staining) nucleolar organizer regions (AgNOR). 

NORs are important for regulating protein synthesis, and  their increase in number from weeks 19  to the 35  of gestation, for example, is indicative of immune system maturation in human fetuses (6,7). NORs are characterized by high polymorphism, as it has been found in 9- to 12-week-old human fetuses  (8), and also by high  variablility in their numbers during human development. In newborns and infants, a relatively high modal number of NORs  is found, whereas in older individuals this number is significantly reduced.  It has been suggested that at a young age due to the obvious enhanced growth and differentiation, more gene sites may be transcriptionally active and show a higher number of AgNORs,  but with advancing age and development, many of these may be gradually repressed or inactivated. 

AgNOR evaluations are performed for processes in which cell proliferation plays an important role, such as embryogenesis.  It has been shown, for example, that gut-associated lymphoid cell proliferation and maturation in 10- to 35-week-old fetuses can be assessed by determination  the number of AgNOR dots (7). NORs have been used to study fetal thymuses (9). It has been shown that type I epitheliocytes (subcapsular-perivascular) of the cortex presents a higher number of AgNORs relative to other cell types between developmental weeks  10 and 15. This reflects their intense protein synthesis, a fact that explains the increased secretion of b2-microglobulin, which releases immature lymphocytes from the yolk sac and liver. A gradual increase in the average number of AgNORs was observed in all thymic epitheliocytes between weeks 10  and  35. This increase might be due to the intense functional activity of all of the epitheliocytes participating in the proliferation, differentiation and issue in the circulation of mature T lymphocytes, which takes place after week 17 of development. The 17-week-old thymus appears fully differentiated, and begins producing the main type of thymocytes from this stage on throughout life.

NORs were used to study of the cellular activity of the embryonal mesenchyme in order to determine the origin of the primitive lymph vessels (9). A statistically significant difference in an average AgNOR numbers in lymph vessels vs. blood vessels was found in the endothelial  and mesenchymal cells  during weeks 10  to 15 of gestation. After week 20, no statistically significant difference was found in this parameter. It has been suggested that development of the lymph vessels follows that of the blood vessels. Furthermore, the intense protein synthesis between weeks 10  and 15 of development is an additional proof for the view that the primitive lymph vessels derive  from clefts into the embryonal mesenchyma and not from capillary offshoots of the blood vessels endothelium.

AgNOR technique was used in experimental teratology to evaluate the causes of fetal limb deformity under maternal administration of retinoic acid (10). It was found that in 15-day-old rat fetuses, hypoplasia and disorientation of hindlimbs are present in 90% of the cases. The histological study showed a reduction in mitotic and NOR activities of mesenchymal cells, an  increase in volume of the vascular lumen,   a reduction in the volume of nerve structures, and a reduction in the percentage of pre-rhabdomyoblastic cells. Somitic NOR activity decreased relative to the control group. This  finding suggests that a particular pathology of the somites  might be involved in clubfoot pathogenesis, and that this pathology is related to a decrease in NOR activity.

We studied of the role of NORs in blast­-transformation of lymphocytes in human fetuses and newborns under local (bronchopneumonia) and generalized (sepsis) microbial in­fections  (3). The material for comparison included three groups of 22- to 42­-week-old fetuses and newborns. The first group (without infections or other antigenic effects) contained 25 fetuses and newborns who died as a result of intranatal asphyxia, respiratory distress syndrome (RDS), hya­line membrane disease of the lungs, or brain hemor­rhage. The second group included 20 fetuses and newborns who died of bronchopneumonia. The third group consisted 27 newborns who died of sepsis. Spleen growth and differentiation in the first group were accompanied by a decrease in the num­ber of follicles per 10,000 μm² and an increase in their area with gestation (Table XVIII). The number of small and medium-sized lympho­cytes/10,000 μm² of follicular area increased whereas the number of lymphoblasts decreased. This resulted in a decrease in the mean nuclear diameter of the lymphoid cells. The mean area of the lymphoid cells and the number of AgNOR/nucleus also decreased. A high negative correlation was found between these parameters and fetal age (r = -0.68, p < 0.01). The number of mitotic cells was very low, 0.36±0.06/10,000 μm², representing 0.14% of total cells. In newborns dur­ing the first week of life, the number of AgNORs/nucleus decreased from 1.64±0.12 to 1.28±0.04 (p < 0.01) and then increased within 2 to 4 weeks to 1.5±0.04 (p < 0.01). These  data exhibit good relationships between some cell parameters and AgNOR features (Table XIX).

Table XVIII. 

Relationship to gestational age in some morphometric and image analysis parameters of lymphoid cells of the splenic follicles in fetuses and infants died without infectious effects (After ref. 3)

^a Mean ± SD.

^b Significantly different from values in young fetuses, p<0.05-0.01.

^c Per 10,000 μm².

Table XIX.

Coefficients of correlation between AgNOR features and some cell parameters

(After ref. 3  )

Table XX.

Effects of infectious antigens on some morphometric and image analysis parameters of lymphoid cells of the splenic follicles in fetuses and infants (After ref.  3)

^a Mean ± SD.

^b Significantly different from values in the first group, p<0.05-0.01.

^c Per 10,000 μm².

The development of an infectious disease such as bron­chopneumonia in the perinatal period is characterized by very active transformation of lymphocytes into lymphoblasts, re­sulting in a significant increase, compared to patients without infections, in the number of lymphoblasts and a significant decrease in the number of lymphocytes (Table XX). The main types of cells were CD20+ cells (B lymphocytes) and CD3+ cells (T lymphocytes). CD45RO+ cells were seen in lower amounts (3). The number of IgM+ cells increased from 0.1-0.5 to 0.9-2.8/10,000 μm². The number of dividing lymphoid cells remained small (1.28±0.24/10,000 μm², or 1.58% of all cells), whereas the number of AgNORs and their area increased significantly. The area of the lymphoid follicles and the number of cells/10,000 μm² decreased, reflecting a decrease in the total number of lymphoid cells in the spleen: from 2936±139/10,000 μm² of follicular area in infants without infection to 1894±54/10,000 μm² in infected infants.

Sepsis causes severe morphological changes in the lym­phoid organs (Table XX). The number of the splenic follicles and their area as well as the number of lymphoid cells in the follicles decreased relative to the group with bronchopneumonia. These changes were reflected in the presence of so-called "bare" central arteries in the spleen. Transfor­mation of lymphocytes into lymphoblasts decreased, and this was manifested by a 25% decrease in the number of lymphoblasts as compared to the number in bronchopneumonia. The number of mi­totic cells decreased from 1.28±0.24 in cases with bronchopneumonia to 0.43±0.06/10,000 μm² with sepsis. The num­ber of AgNORs/nucleus decreased and their area tended to­ward a decrease. Some dystrophic and necrotic changes were found in lymphocytes. A good correlation was found between the number of AgNORs and the number of lymphoid cells (r = 0.67 and 0.85), as well as between the former and the rate of mitosis (r = 0.97).

The development of lymphoid organs (growth, differentiation and matura­tion) in 22- to 42-week-old fetuses is accom­panied by an increase in the relative number of small and medium lymphocytes and by a decrease in the relative num­ber of lymphoblasts (1,2). The percentage of lymphocytes in fetal white blood cells is 79% in early second- and third-trimester fetuses, but decreases to 40% at birth (11).   Age-related changes have been found in cytokine production, immunoproliferative T-lymphocyte response and NK cell activity in newborns, children and adults (12). After birth, alterations in some of the morphological and mor­phometric parameters of the splenic cells and their AgNORs were seen even in uninfected newborns (2,3). The changes were usu­ally weak and are overshdowed by the high antigenic effects that accompanying physiological contamination of the infant after birth, especially as related to infections.

The development of infection causes considerable changes in the infant's lymphoid system, and the main manifestation of this process is the transformation of lym­phocytes into lymphoblasts (1,2). The number of lym­phoblasts in bronchopneumonia increased to up 32.85±3.11/10,000 μm², as compared to 5.69±0.48/10,000 μm² in infants who died of noninfectious diseases. The num­ber of mitoses per 10,000 μm² in the lymphoid cells of infants who died of bronchopneumonia increased to 1.28±0.24, as compared to 0.36±0.06 in children who died of non-infectious diseases. This may explain the absence of germinative centers in the splenic lym­phatic follicles. An increase in AgNOR numbers also accompanies the process of lym­phocyte transformation to lymphoblasts in newborns sub­jected to infectious influences (3).

Both fetuses infected with cytomegalovirus,  rubella and toxoplasmosis and their mothers have significant identifiable changes in white cell counts and T-lymphocyte subpopulations compared to controls   (13). The percentage  of CD3⁺ and CD8⁺ lymphocytes was significantly higher in infected mothers compared to controls, while the percentage of CD19⁺  lymphocytes and the CD4⁺/CD8⁺ ratio were lower. Infected mothers carrying infected fetuses had significantly lower white blood cell counts compared to those infected mothers without fetal infection. The percentage of CD3⁺ T lymphocytes was significantly higher and the CD4⁺/CD8⁺ ratio lower in infected fetuses compared to controls and noninfected fetuses in infected mothers. This is in line with observations relating to an extreme increase in RNA and protein synthesis, which was found to parallel the activation of the lymph system during immune reaction (14,15).

In the presence of severe infections such as sepsis, the size and number of AgNORs per nucleus decrease sig­nificantly, reflecting inhibition of the infant's immune system. This process is thought to be a manifestation of the general decompensation of the lym­phoid system which develops as a result of severe antigenic ef­fects. In newborns who died of sepsis, there was an almost 15-fold decrease in splenic lymphatic cells as compared to unaffected infants. It has been suggested  that in new­borns suffering from bronchopneumonia there was already a significant decrease in the number of these cells, including lymphoblasts, and that this process just became more severe in sepsis  (3).

The spleen is the main organ of the immune reaction in fetuses and newborns, and its follicles are the main site of lymphoid cell concentration. The disastrous decrease in the number of these cells reflects an inadequacy of the new­born's immune response to the severe infections. A possible explanation for this is that an in­creases in the number of lymphoblasts in fetuses and new­borns occurs not only by mitotic division, as in children and adults, but also by transformation of lym­phocytes to lymphoblasts (16). This results in the rapid depletion of the total amount of lymphatic cells in the organism.

The generalized devastation of lymphoid organs is the ma­jor pathological process  reflecting the exorbitant antigenic effects on a developing organism under sepsis. In sep­ticemia, for example, the more common type of sepsis in the perinatal period, this devastation is the basis for pathomorpho­logical diagnosis of the disease. A similar picture charac­terizes the Rh-HDN or severe in­flammatory processes in pregnant mothers (17). A high correlation between the changes in NORs in lymphocytes and lymphoblasts and the state of the lym­phoid organs in sick infants suggests that these changes occur not only in the lymphocytes of the splenic follicles but also in those of the peripheral blood. 

Changes in NORs and nuclei of the splenic lym­phoid cells may reflect their role in the immune reaction of fetuses and new­borns to infections such as bronchopneumonia and sepsis. These data agree with the pathomorphological picture and reflect a close relationship between NORs and the synthesis of DNA, RNA and proteins in cells (5). The changes in lymphoid cell NORs are connected with these cells' reaction to infectious antigenic effects.  NORs changes can be used as a reliable parameter in diagnostic practice for evaluating the immune reactive process in newborns.

 9.2)  Intraorganic immunity of fetuses and newborns  with infectious diseases

At least two types of IgA circulat­ing in the blood and lymph are recognized: serum IgA and the so-called secretory IgA (sIgA). The latter is secreted by cells within mucosal membranes, and is more important than sIgG or sIgM (18). sIgA is considered to be an important part of an organism's protective mechan­ism against penetration of infection through the mucosal membranes of the respiratory and gastrointestinal tracts, and urogenital organs.

In fetuses, IgG and IgA responses are expressed by B cells (19,20). T helper cells are present and functional, but their capacity to drive IgG and IgA responses is impaired.  Development of clonal diversity for both T and B cells begins during the first trimester of human gestation and is far advanced by mid-gestation (21). It has been well documented that in normal human intrauterine development, IgA increases on the syncytial trophoblast at 8 to 10 weeks of gestation; IgE is present on the surface of the trophoblast only during spontaneous labor (22); and IgG is observed on the syncytial trophoblastic cell membrane and basement membrane and in the cytoplasm and nuclei at all stages of gestation (23,24).  The detection of the sIgA in the amniotic fluid  in the early periods of pregnancy leads to the assumption  of IgA in the amniotic membranes (25). The presence of IgA in the chorion and decidua suggests that the placenta is  a first barrier against infection of the amniotic cavity (26,27).

In addition to the predominant maternal IgG, the amniotic fluid contains different molecular forms of fetal Igs. These function as an immune barrier against infection and against mother-derived autoantibodies. In studying the molecular status of antibodies in the human amniotic fluid, IgG is found to be the major isotype, IgA is much less abundant, and IgM is not detected at all (21). IgA is monomeric, with a low level of sIgA and with various amounts of free SC. The presence of a low level of SC-containing Igs of small size is confirmed during the last trimester of pregnancy.

Abnormal fetal development caused by infections or inflammation is characterized by a high blood concentration of different types of Igs (28,29), but this is not the case with antigenic effects (30). Several types of autoantibodies can be found in some sera of newborns, and increased IgM concentration may reflect a polyclonal antibody response (31). Cytomegalovirus infection or Toxoplasma gondii cause the appearance of IgM antibodies in the fetal blood (32,33).   Neospora  caninum-specific fetal IgG and IgM antibodies were detected in cattle inoculated with N. caninum at mid-gestation (34).  Elevated IgA and IgM levels have been found in abnormalities of the central nervous system and in congenital malformations (35,36). 

Amniotic fluid levels of sIgA increase significantly during normal pregnancy (21,37). sIgA has been estimated in the amniotic fluid of the third trimester of pregnancy and in mucus samples of pharyngeal cavities and urine of newborns to test the hypothesis that there is a connection between sIgA content in the amniotic fluid and fetal pulmonary maturity (38). Small amounts of SC  and only a few IgM-, IgD- and IgG-producing cells were present in the tracheal surface and gland epithelium during the fetal period and increased towards term, but no IgA- or IgE-producing cells were found (39). These features probably reflect local activation of the immune system in response to environmental factors.

In fetuses with prematurely ruptured membranes, an increase in  the concentration of  IgA has been found in the chorioamniotic membrane  (40). Preterm premature rupture of membranes and microbial invasion of the amniotic cavity are associated with a robust host inflammatory response in the fetal, amniotic, and maternal compartments (41). IL18, a proinflammatory pleiotropic cytokine that has been implicated in the host defense against infection, increased in cases of the microbial invasion of the amniotic cavity (42). 

In the  premature infants who were either stillborn or died shortly after delivery (gestational age 24-32 weeks), in full-term infants who died during the first 3  weeks after birth, and in infants who died in the postneonatal period,  only a few IgM- and IgG-producing cells were present in the duodenal mucosa throughout the period studied, while no IgA immunocytes were seen before the first week after birth (39).  The appearance of IgA immunocytes suggests that the intestinal immune system is modulated in response to environmental factors shortly after birth.

Among fetal infections, candidal chorioamnionitis is an uncommon and apparently rather indolent intrauterine infection in which the fetus is able to marshal some of the immunological forces at its disposal against an easily visualized antigen impinging on lung mucosal surfaces (43). Chorioamnionitis was associated with an intrauterine inflammatory response of the fetal lung characterized by a severe infiltration of macrophages, neutrophils, and lymphocytes as well as  by increased expression of IL8 mRNA (44). Apoptosis and proliferation are important features of chorioamnionitis-associated lung injury: chorioamnionitis induces apoptosis of distal airway epithelial cells via the caspase-8 pathway and interferes with the normal proliferative activity of epithelial, endothelial, and smooth muscle cells in fetal lungs (45).

The inflammatory response in candidal chorioamnionitis is manifested in the Ig-containing lesions, which probably originated in dense-staining plasmacytoid and immunoblastic cells in the inflammatory infiltrates (46,47). The finding of giant cell pneumonitis suggests that the fetus can mount a brisk inflammatory and immune response at as early as 18 weeks of gestation and that mucosal exposure to this antigen can result in IgA production by the lungs. A similarly high amount of specific T. gondii IgA antibody has been found in cord blood and in neonatal blood,  64% and 66%, respectively  (48).

The presence of granular deposits of Igs within the vessel walls with acute atherosis may be related to an immunological disorder, probably mediated by immune complexes. Acute atherosis associated with human fetal growth restriction (FGR) is manifested in massive intramural deposits of IgM, and slight deposits of IgA and IgG (49,50). No intramural deposition of Igs or complement has been observed in vessels with or without physiological changes. In severe pregnancy-induced hypertension, there are depositions of immunocomplex and complement on the vessel walls of the chorionic villi and decidua (51,52). Immunological factors play an important role in the development of this disorder: the positive expression rates of the IgA, IgG, IgM and C3 in the vascular wall were significantly higher than those in normal-term pregnancies. Patients with severe vessel lesions had a significantly greater incidence of fetal loss than those with only mild to moderate lesions.

Evaluation of the maternal-fetal interface reveals an increased deposition of Igs  that may be associated with a common antigen as an immunological etiology for preeclampsia (53). Antiphospholipid antibodies, for example, may play a pathogenic role in some cases of preeclampsia: elevated levels of IgG or IgM to cardiolipin and phosphatidylserine were detected in 11% of women with preeclampsia in the third trimester, compared to only 3% in controls (54). 

Chorioamnionitis is considered an important risk factor for early-onset infection in premature newborns. Septicemia, pneumonia or omphalitis were documented in 20% of infected premature newborns, and inflammatory lesions in the placenta were observed in all of them (55).  The probability of neonatal infection in premature newborns was 62.5% when polymorphonuclear neutrophils were present in the chorion and amniotic membrane, as compared to 0.5% when these tissues were normal.

The frequency of clinical chorioamnionitis in preterm premature rupture of the fetal membranes increases with the duration of the interval between membrane rupture and delivery (56). The prevalence and severity of pathological evidence of intrauterine infection is also correlated with the interval between membrane rupture and delivery. The amount of IgA in the chorioamniotic membrane was 24.58 mg/dl in patients whose membranes had been ruptured for longer than 10 h, as compared to 2.52 mg/dl in membranes which had been ruptured for less than 10 h (40). These data indicate that the increasing IgA in patients after 10 h of latency probably represents the beginning of an ascending colonization of bacteria which could be a source of the impending infection.

A characterization of the fetal-derived inflammatory cell reaction may be important in understanding of both the intrauterine and the antenatal immunological response of the neonates to viral infection. The marked hyperplasia of fetal-derived placental macrophages (Hofbauer cells) is considered an example of the immunological features of the fetal inflammatory response to placental cytomegalovirus infection (57,58). Lymphocytic villitis is characterized by the presence of T-cell and not B-cell antibodies.   The plasmacellular villitis  contains both IgG- and IgM-secreting cells at as early as the second trimester of gestation. No IgA-positive plasma cells are observed.  CD3⁺ lymphocytes predominated in syphilitic villitis, with slightly more CD8⁺ cells than CD4⁺ cells (59). CD68 and HLA-DR-positive cells are as frequent as CD3⁺ cells, but B-lymphocytes are rare.

Neither serum IgM  nor IgA or sIgA cross the placenta (60). In fetuses, the serum IgA is formed in the presence of perinatal infections (61) and other antigenic effects such as HDN (17). A newborn receives sIgA with the colostrum and maternal milk (62). The sIgA plays an important role in the development of local immune reactions in the gastrointestinal tract normalizing microbiocenosis (63). This is expressed by an increased amount of IgA-­secreting cells and SC synthesis (39). The high importance of sIgA in children is reflected in the fact that the synthesis of the child form of sIgA and its movement through the mucosal membranes increase sharply during the first three months after birth  and it reaches adult levels in 2-year-olds (64).   

The distribution and functional activity of sIgA in mucosal membranes and lymphoid organs were studied in full-term and LBW fetuses and newborns (65). Thirty-eight fetuses and newborns were divided into three groups. The first group (without infections) contained 15 fetuses and newborns who had died as a result of non-antigen-induced diseases, such as intranatal asphyxia, RDS, or brain hemorrhage. The second group included 10 fetuses and newborns who had died of bronchopneumonia. In the third group, 13 fetuses and newborns were included who had died of sepsis.

In fetuses from the first group (20 to 21 weeks of gestation),  IgA was found in two types of cells: one type presented B lymphocytes of the spleen and lymph nodes; the other presented the epithelium of the trachea and bronchi, their submucosal glands, and the epithelium of hepatic bile ducts. This IgA is excreted into the lumina of the bronchi and bile ducts, and is, therefore, considered to be sIgA. The number of IgA-secreting epithelial cells varied in the organs studied from 2 to 8 cells/10,000 μm², i.e., from 11% to 26% of the total number of epithelial cells on a slide.

The number of IgA-secreting cells increased with the time of gestation. sIgA was not found in the epithelium or lumina of bronchioles, or in the epithelium of the pancreas or renal pelvis. Secretory IgM and IgG were also not found in any of these organs during gestation. However, synthesis of IgM in the spleen proceeded at a higher rate than that of IgA, especially in the presence of antigenic effects. Maternal sIgG enters the fetus with the amniotic fluid from the 18th week of gestation (66). The absence of sIgM and sIgG shows that in newborns sIgA plays a more pronounced role than other types of Igs. Infection caused a significant decrease in the number of sIgA-containing epithelial cells (Table XXI). In the bronchi, for example, their number decreased from 30% in controls to 18% in newborns who had died of broncopneumonia and to 14% in infants who had died of sepsis. In the intrahepatic bile ducts, these cells numbered 48%, 36% and 14%, respectively. At the same time, the number of IgA-containing granules in the lumina of these organs increased significantly, showing that infection intensifies sIgA secretion.

Table XXI.

The number of IgM- and IgA-containing lymphocytes and epithelial cells in fetuses and newborns with different antigenic and non-antigenic effects (After ref. 65)

  ^a Groups of patients: I, died without antigenic effects; II, died of bronchopneumonia;                     III, died of sepsis.

  ^b Mean ± SE.

  ^c Significance different from group 1, p<0.05-0.01.

  ^d Significance different from group 2, p<0.05-0.01.

sIgA participates in different immune pro­cesses, such as inhibition of microbial adherence, antigen exclusion, virus and toxin neutralization, and modula­tion of enzyme activity (67). Fixing on receptors for Fc, fragments of macrophages (18) and neutrophils (68), sIgA may participate in phagocytosis, cause the degranulation of eosinophils (69), provide an alternative way of fixing complement (70), and may participate in antibody-dependent cytotoxicity. 

In the presence of infection, the number of sIgA-containing epithelial cells decreased, while the number of IgA-positive lymphocytes in the spleen increased, reflecting the increased synthesis of IgA in this organ (65). The low and inverse correlation between the number of IgA-synthe­sized lymphocytes and sIgA-containing cells in the epithelium of bronchi (r = -0.34) and bile ducts (r = -0.31) can be explained in several different ways. First, the synthesis of serum IgA and sIgA originates from separate pools of B lymphocytes, and therefore, their contents do not overlap (64).  Secondly, although the spleen is the main organ of the lymph system in fetuses and newborns, IgA synthesis occurs in other organs as well, such as the liver, lymph nodes and lymph nodules of the gastrointestinal tract. Finally, a decrease in the number of IgA-positive epithelial cells may be caused by the rapid exhaustion of their SC, the cellular amount of which decreases as a result of IgA secretion into the organ lumina. This last phenomenon can be considered a manifestation of the 'immaturity' of the immune system and its consequent rapid exhaustion under even weak antigenic effects (1,2).

The presence of sIgA in the mucosal membranes of the trachea, bronchi and intrahepatic bile ducts is related to their importance in protecting against physiological contamination by microbes after birth and in preventing inflammatory processes. The presence of a large amount of sIgA in mid-gestation fetuses (20-21 weeks), is considered to be evidence of early maturation of the immune system, or at least this component of it.

              9.3) Insufficiency of the immune system in fetuses and infants

                                                  with pneumonia and sepsis

In higher vertebrates, the ability to respond to antigen develops in a slow, controlled, stepwise fashion as a function of ontogeny. The process takes months in humans and lambs, and days to weeks in mice. In humans, the ability to mount an effective humoral response to antigens, including pathogenic bacteria and vaccines, develops in a sequential fashion and is not fully mature until well after infancy (71).  The delay in the ability to respond to specific antigens increases young infant's susceptibility to infection, particularly those that are born prematurely. The capacity of lymphocytes to generate a heterogeneous repertoire of antigen-binding receptors lies at the heart of their ability to mount a specific humoral response to diverse antigens. Antibody repertoire development appears to be endogenously controlled and adheres to an individualized developmental progression that probably contributes to the relative immaturity of the neonatal immune response (72). The reasons underlying constraint of the antibody repertoire in the first and second trimester,  and its slow developmental progression during the third trimester and early infancy, remain a mystery.

An increase in the synthesis of IgG, IgA and IgM immunoglobulins is one of the characteristics of defense mechanisms which protect fetuses against infection. Infants with congenital toxoplasmosis, for example, show evidence of increased intrauterine IgM and IgA synthesis (73,74). In the serum of newborns and infants born to mothers suffering from different viral and parasitic disease (rubella virus, cytomegalus virus, Listeria monocytogenes, Chlamydia trachomatis and T. gondii), IgA and IgM were found at very high levels, significantly higher than in controls samples (75-77).

Inflammatory diseases and, especially, sepsis can cause infant mortality, premature birth, and LBW  infants. The peculiarities of the immune-response mechanism and its relation to pathogenesis in neonates is generally described only as "immature and naive" (78). Thus, the well-known susceptibility of LBW infants to infections is blamed on deficiencies in cellular and humoral immunological mecha­nisms. Fetal immune incompetence is considered to be a major reason for the high death rate of infants or for retardation of their postnatal growth and development (79). Different parts of the immune system in such infants show signs of underdevelopment, yet  little information exists on the possible contribution of the pathology of immune organs to the birth and death of LBW neonates.

Lymphoid organs develop in a controlled, stepwise fashion during ontogeny (80). Organization of the primary structures is not complete until the end of the second trimester. Expression of an expanded repertoire could be deleterious to an infant with a disorganized lymphoid system; expression of the conserved fetal repertoire may play an important protective role, or expression of a "mature" repertoire could be deleterious to the developing infant or to the fetal-maternal balance. Clearly, however, processes critical to the establishment of a mature repertoire are active and changing during the third trimester of gestation. The appearance of secondary structures that represent a reaction to antigen in the primary lymphoid nodules, i.e., follicles, is first observed at 30 weeks of gestation (2).  Antigenic effects, such as neonatal sepsis and chorioamnionitis, induce morphological modifications and shrinkage of the lymphoid organs, particularly the thymus (81). Fetuses with chorioamnionitis or neonatal sepsis show spleen-cell depletion, involving both B and T lymphocytes.

In diseases based on bacterial or viral infections, B and T lymphocytes play an essential role, which is reflected by their percentages in the blood. In infants with acute upper respiratory tract infections, for example, percentages of B lymphocytes in the peripheral blood are markedly increased (82,83). Early intrauterine rubella  infection   has a profound effect on the developing immune system in fetuses that is manifested in complete immune paralysis, Ig abnormalities, and loss of antibodies to rubella (84).  These defects are transient, but the absence of IgA may be permanent. No such defects have been observed in other congenital viral infections, but precocious development of Igs and germinal follicles does occur.

Neonatal bacterial sepsis is often characterized by a fulminate clinical course and highly elevated plasma levels of proinflammatory cytokines (85). Activation of cord blood cells by infectious stimuli, such as  Streptococcus agalactiae, is comparable to the adult immune response in terms of expression of proinflammatory cytokines.  The malaria parasite Plasmodium falciparum caused an increase in the synthesis of Igs,  especially of IgG,  the concentration of which in the cord blood was 69%, as compared to 6% and 4.4% IgM and IgE, respectively (86). Neonates born to malaria-positive mothers mounted predominantly Th2-type immune responses. It appears that neonates born to malaria-infected mothers may relatively be high susceptible to malaria attack during the first years of life.

The immune response to microbial effects in fetuses and newborns with pneumonia is manifested in specific differences in the morphological features compared to those seen in children and adults. Such an immune response has been termed "immune insufficiency" (1,2). The role of the lymphoid system in fetuses and neonates who succumb to severe antigenic effects, such as pneumonia and sepsis, has been studied using morphological, morphometric and immunohistochemical analyses (87).

In the aforementioned study of three groups of fetuses and newborns - those who died of non-antigen-induced diseases (intranatal asphyxia, RDS, or brain hemorrhage), those who died mainly of bronchopneumonia and alveolitis-pneumonia accompanied by RDS, hyaline membrane disease and inborn heart disease, and those who died with sepsis - it was found that in the absence of antigenic effects, the lymphoid organs (thymus, spleen and lymph nodes) are morphologically formed by 22 to 24 weeks of gestation. The thymus consists of a cortex, medulla, and thymic corpuscles (Table XXII). The spleen consists of white and red pulps with differentiation of follicles and periarterial lymphoid sheaths (PALS) in the white pulp (Table XXIII). Follicles are quite pronounced in the lymph nodes.

When the microbial antigenic effects are mild (e.g., in alveolitis-pneumonia), the immune reaction is generalized and spreads to all of the lymphoid organs. It is manifested in an increase in the number of CD20⁺ B lympho­blasts and fewer small lymphocytes, as a result of their acti­vation (blast transformation) (Table XXIII). There are high amounts of T lymphocytes in the PALS, IgM and IgA-synthesized cells and a high number of macrophages and dendritic cells (65). The accidental involution (AI) of the thymus reaches the third phase, as reflected by a significant increase in the number of thymic corpuscules (Table XXIV). These changes are different from those described for chil­dren and adults. The germinal centers of the follicles, massive apoptosis of lymphocytes, multiplication of lympho­blasts and the formation of mature plasmocytes, which are characteristic in children and adults, are absent in infants (1,2).

Table XXI.

Morphometric parameters of the thymus in different groups of fetuses

(mean±SE) (After ref. 87)

^a As a percentage of the total area of the whole slide.

^b Significantly different from group 1, p<0.02-0.01.

^c Significantly different from group 2, p<0.05-0.01.

Table XXII.

Morphometric parameters of the spleen in different groups of fetuses

(mean±SE) (After ref. 87)

^a-c See footnotes to Table XXII.

Table XXIV.

Area of thymic corpuscules in fetuses and infants (as a percentage of the total area of the whole slide) (After ref. 87)

^a Significantly different from group 1, p<0.01.

^b Significantly different from other infants in the same group, p<0.01.

^c Significantly different from infants in other groups, p<0.01.

The morphological features of immune insufficiency in infants can be displayed even under mild antigenic effects: the number of cells in the splenic follicles decreases by 30%, and the area of the parenchyma of lymph nodes de­creases by 21 %. In LBW neonates, the thymus is characterized only by the first phase of AI and by weak proliferation of the reticular epithelium. All of these data characterize the immune re­sponse of fetuses and newborns to antigenic effects as a special form of the fetal type (1,2).   

Among fetuses and newborns with bronchopneumonia, the lymphoid organs showed changes reflecting a marked generalized immune response (87). The number of CD20⁺ lymphoblasts in the spleen and lymph nodes is four to eight times higher and the number of lymphocytes, especially B cells,  in the splenic follicles is significantly lower compared to counterparts without antigenic effects. The ger­minal centers in the follicles are not formed, the number of mitotic and apoptotic cells is low, and the mature plasma cells are absent. There is an increase in the number of CD3⁺ T lymphocytes in the PALS, dendritic cells in the follicles of the white pulp, and of macrophages in the red pulp of the spleen  (Table  XXIII). There is a significant decrease in the number of follicles in the spleen (per mm²), and in the number of cells and the parenchyma of the lymph nodes. In the thymus, both AI and devel­opment of the thymic corpuscles are correlated with age: AI in the second and third phases is seen only in full-term neonates or in LBW infants who died after the second week of life  (1,2).   

In the group with sepsis, there are fewer proliferative and many more destructive changes in the lym­phoid organs (87). The spleen and lymph nodes demonstrate  blast-transformation of CD20⁺ B lympho­cytes and an increase in the number of macrophages and dendritic cells. There is a sharp decrease in the number of lymphoid cells, which is especially noticeable in T and B lymphocytes  and IgM-containing cells. The number of follicles is one-third that in fetuses without antigenic effects, and their cellular area is reduced by half. In the spleen, which is the main immune organ in fetuses and newborns, the number of lymphoid cells decreases by 60% to 75% compared with their number in neonates without antigenic effects. A particularly sharp decrease in the total number of lymphoid cells is seen in LBW infants in whom the entire mass of the lymphoid organs and, particu­larly, the spleen is five to seven times smaller than in full-term infants (1). Devastation of the cortex of the thymus and of other lymphoid organs is found in the most severely affected cases.  The third phase of AI iss found in the thymus. The area of the thymic corpuscles increases significantly (Table XXIV) and persists of "pearls" from the horn-like epithelium.  The marked proliferation of thymic cor­puscles in these cases can be considered an adaptive re­action of infants to an abnormal situation. 

The morphological signs of decompensation of the lym­phoid system afflicted by severe antigenic effects have been observed in cases in which the lymph system was incapable of providing an adequate immune response. This inability can develop under mild antigenic effects in parallel with the so-called immune incompetence or immunodeficiency, seen in LBW infants with immaturity or genetic immunodeficient syndrome  (88). Unfortunately, the terms of immune incompetence or immunodeficiency describe only insufficiency of the immune system and cannot be considered a phenomenon of decompensation. The latter can develop under extremely severe antigenic microbial ex­posure and manifests itself in intoxication and dysfunction of different organs and systems. Decompensation has also been observed in fetuses under massive non-microbial anti­genic effects, consequences of the edemic form of HDN or preeclampsia (2). However, the morphological features of sepsis described herein, and es­pecially the increase in the number of neutrophils, did not develop under these conditions.

It can be suggested that sepsis in infants is a result of generalized decompensation of the lymphoid system, especially of its B and T parts.  Participation of neutrophils and eosinophils in the  response to sepsis  can be considered a manifestation of an organism's compensatory reaction, an additional mechanism of an antimicrobial defense. This reaction increases in parallel to the advance of gestation and postnatal age (1). 

There are three possible causes of decompensation of the lymphoid systems in sepsis: i) invasion by even a small amount of highly pathogenic microbes, ii) massive infection by moderately pathogenic microbes, and iii) inadequacy of the immune system, or immune incompetence. This last param­eter is a common reason for the development of sepsis in infants. The process begins locally, and eventually the entire immune system exhibits features of decompensation, such as underdevelopment of the thymus, of T and B lymphocytes, neutrophils, and macrophages (89). A similar picture is observed in pre-term LBW infants (90,91). The low weight of the lymphoid organs is also relevant to the development of immune incompetence, because such organs become exhausted under even weak antigenic affects.

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