Epithelial-mesenchymal transition in oral squamous carcinoma cells

In epithelial-mesenchymal transition (EMT), epithelial cells acquire traits typical for mesenchymal cells, dissociate their cell-cell junctions and gain the ability to migrate. EMT is essential during embryogenesis, but may also mediate cancer progression. Basement membranes are sheets of extracellular matrix that support epithelial cells. They have a major role in maintaining the epithelial phenotype and, in cancer, preventing cell migration, invasion and metastasis. Laminins are the main components of basement membranes and may actively contribute to malignancy. We first evaluated the differences between cell lines obtained from oral squamous cell carcinoma and its recurrence. As the results indicated a change from epithelial to fibroblastoid morphology, E-cadherin to N-cadherin switch, and change in expression of cytokeratins to vimentin intermediate filaments, we concluded that these cells had undergone EMT. We further induced EMT in primary tumour cells to gain knowledge of the effects of transcription factor Snail in this cell model. The E-cadherin repressors responsible for the EMT in these cells were ZEB-1, ZEB-2 and Snail, and ectopic expression of Snail was able to augment the levels of ZEB-1 and ZEB-2. We produced and characterized two monoclonal antibodies that specifically recognized Snail in cell lines and patient samples. By immunohistochemistry, Snail protein was found in mesenchymal tissues during mouse embryonal development, in fibroblastoid cells of healing skin wounds and in fibromatosis and sarcoma specimens. Furthermore, Snail localized to the stroma and borders of tumour cell islands in colon adenocarcinoma, and in laryngeal and cervical squamous cell carcinomas. Immunofluorescence labellings, immunoprecipitations and Northern and Western blots showed that EMT induced a progressive downregulation of laminin-332 and laminin-511 and, on the other hand, an induction of mesenchymal laminin-411. Chromatin immunoprecipitation revealed that Snail could directly bind upstream to the transcription start sites of both laminin α5 and α4 chain genes, thus regulating their expression. The levels of integrin α6β4, a receptor for laminin-332, as well as the hemidesmosomal complex proteins HD1/plectin and BP180 were downregulated in EMT-experienced cells. The expression of Lutheran glycoprotein, a specific receptor for laminin-511, was diminished, whereas the levels of integrins α6β1 and α1β1 and integrin-linked kinase were increased. In quantitative cell adhesion assays, the cells adhered potently to laminin-511 and fibronectin, but only marginally to laminin-411. Western blots and immunoprecipitations indicated that laminin-411 bound to fibronectin and could compromise cell adhesion to fibronectin in a dose-dependent manner. EMT induced a highly migratory and invasive tendency in oral squamous carcinoma cells. Actin-based adhesion and invasion structures, podosomes and invadopodia, were detected in the basal cell membranes of primary tumour and spontaneously transformed cancer cells, respectively. Immunofluorescence labellings showed marked differences in their morphology, as podosomes organized a ring structure with HD1/plectin, αII-spectrin, talin, focal adhesion kinase and pacsin 2 around the core filled with actin, cortactin, vinculin and filamin A. Invadopodia had no division between ring and core and failed to organize the ring proteins, but instead assembled tail-like, narrow actin cables that showed a talin-tensin switch. Time-lapse live-cell imaging indicated that both podosomes and invadopodia were long-lived entities, but the tails of invadopodia vigorously propelled in the cytoplasm and were occasionally released from the cell membrane. Invadopodia could also be externalized outside the cytoplasm, where they still retained the ability to degrade matrix. In 3D confocal imaging combined with in situ gelatin zymography, the podosomes of primary tumour cells were large, cylindrical structures that increased in time, whereas the invadopodia in EMT-driven cells were smaller, but more numerous and degraded the underlying matrix in significantly larger amounts. Fluorescence recovery after photobleaching revealed that the substructures of podosomes were replenished more rapidly with new molecules than those of invadopodia. Overall, our results indicate that EMT has a major effect on the transcription and synthesis of both intra- and extracellular proteins, including laminins and their receptors, and on the structure and dynamics of oral squamous carcinoma cells.

Role of Basement Membrane and Extracellular Matrix Proteins in the Adhesion and Spreading of Immortalized Human Corneal Epithelial Cells

The repair of corneal wounds requires both epithelial cell adhesion and migration. Basement membrane (BM) and extracellular matrix (ECM) proteins function in these processes via integrin and non-integrin receptors. We have studied the adhesion, spreading and migration of immortalized human corneal epithelial (HCE) cells and their interactions with the laminins (Lms), fibronectins and tenascins produced. Human corneal BM expresses Lms-332 and -511, while Lm-111 was not found in these experiments. HCE cells produced both processed and unprocessed Lm-332, whereas neither Lm-111 nor Lm-511 was produced. Because HCE cells did not produce Lm-511, although it was present in corneal BM, we suggest that Lm-511 is produced by stromal keratocytes. The adhesion of HCE cells to Lms-111, -332 and -511 was studied first by determining the receptor composition of HCE cells and then by using quantitative cell adhesion assays. Immunofluorescence studies revealed the presence of integrin α2, α3, α6, β1 and β4 subunits. Among the non-integrin receptors, Lutheran (Lu) was found on adhering HCE cells. The cells adhered via integrin α3β1 to both purified human Lms-332 and -511 as well as to endogenous Lm-332. However, only integrin β1 subunit functioned in HCE cell adhesion to mouse Lm-111. The adhesion of HCE cells to Lm-511 was also mediated by Lu. Since Lm-511 did not induce Lu into focal adhesions in HCE cells, we suggest that Lm-511 serves as an ECM ligand enabling cell motility. HCE cells produced extradomain-A fibronectin, oncofetal fibronectin and tenascin-C (Tn-C), which are also found during corneal wound healing. Monoclonal antibodies (MAbs) against integrins α5β1 and αvβ6 as well as the arginine-glycine-aspartic acid (RGD) peptide inhibited the adhesion of HCE cells to fibronectin. Although the cells did not adhere to Tn-C, they adhered to the fibronectin/Tn-C coat and were then more efficiently inhibited by the function-blocking MAbs and RGD peptide. During the early adhesion, HCE cells codeposited Lm-332 and the large subunit of tenascin-C (Tn-CL) beneath the cells via the Golgi apparatus and microtubules. Integrin β4 subunit, which is a hemidesmosomal component, did not mediate the early adhesion of HCE cells to Lm-332 or Lm-332/Tn-C. Based on these results, we suggest that the adhesion of HCE cells is initiated by Lm-332 and modulated by Tn-CL, as it has been reported to prevent the assembly of hemidesmosomes. Thereby, Tn-CL functions in the motility of HCE cells during wound healing. The different distribution of processed and unprocessed Lm-332 in adhering, spreading and migrating HCE cells suggests a distinct role for these isoforms. We conclude that the processed Lm-332 functions in cell adhesion, whereas the unprocessed Lm-332 participates in cell spreading and migration.

The scientific basis and development of a matrix metalloproteinase (MMP) -8 specific chair-side test for monitoring of periodontal health and disease from gingival crevicular fluid

Matrix metalloproteinase (MMP) -8, collagenase-2, is a key mediator of irreversible tissue destruction in chronic periodontitis and detectable in gingival crevicular fluid (GCF). MMP-8 mostly originates from neutrophil leukocytes, the first line of defence cells which exist abundantly in GCF, especially in inflammation. MMP-8 is capable of degrading almost all extra-cellular matrix and basement membrane components and is especially efficient against type I collagen. Thus the expression of MMP-8 in GCF could be valuable in monitoring the activity of periodontitis and possibly offers a diagnostic means to predict progression of periodontitis. In this study the value of MMP-8 detection from GCF in monitoring of periodontal health and disease was evaluated with special reference to its ability to differentiate periodontal health and different disease states of the periodontium and to recognise the progression of periodontitis, i.e. active sites. For chair-side detection of MMP-8 from the GCF or peri-implant sulcus fluid (PISF) samples, a dip-stick test based on immunochromatography involving two monoclonal antibodies was developed. The immunoassay for the detection of MMP-8 from GCF was found to be more suitable for monitoring of periodontitis than detection of GCF elastase concentration or activity. Periodontally healthy subjects and individuals suffering of gingivitis or of periodontitis could be differentiated by means of GCF MMP-8 levels and dipstick testing when the positive threshold value of the MMP-8 chair-side test was set at 1000 µg/l. MMP-8 dipstick test results from periodontally healthy and from subjects with gingivitis were mainly negative while periodontitis patients sites with deep pockets ( 5 mm) and which were bleeding on probing were most often test positive. Periodontitis patients GCF MMP-8 levels decreased with hygiene phase periodontal treatment (scaling and root planing, SRP) and even reduced during the three month maintenance phase. A decrease in GCF MMP-8 levels could be monitored with the MMP-8 test. Agreement between the test stick and the quantitative assay was very good (κ = 0.81) and the test provided a baseline sensitivity of 0.83 and specificity of 0.96. During the 12-month longitudinal maintenance phase, periodontitis patients progressing sites (sites with an increase in attachment loss ≥ 2 mm during the maintenance phase) had elevated GCF MMP-8 levels compared with stable sites. General mean MMP-8 concentrations in smokers (S) sites were lower than in non-smokers (NS) sites but in progressing S and NS sites concentrations were at an equal level. Sites with exceptionally and repeatedly elevated MMP-8 concentrations during the maintenance phase were clustered in smoking patients with poor response to SRP (refractory patients). These sites especially were identified by the MMP-8 test. Subgingival plaque samples from periodontitis patients deep periodontal pockets were examined by polymerase chain reaction (PCR) to find out if periodontal lesions may serve as a niche for Chlamydia pneumoniae. Findings were compared with the clinical periodontal parameters and GCF MMP-8 levels to determine the correlation with periodontal status. Traces of C. pneumoniae were identified from one periodontitis patient s pooled subgingival plaque sample by means of PCR. After periodontal treatment (SRP) the sample was negative for C. pneumoniae. Clinical parameters or biomarkers (MMP-8) of the patient with the positive C. pneumoniae finding did not differ from other study patients. In this study it was concluded that MMP-8 concentrations in GCF of sites from periodontally healthy individuals, subjects with gingivitis or with periodontitis are at different levels. The cut-off value of the developed MMP-8 test is at an optimal level to differentiate between these conditions and can possibly be utilised in identification of individuals at the risk of the transition of gingivitis to periodontitis. In periodontitis patients, repeatedly elevated GCF MMP-8 concentrations may indicate sites at risk of progression of periodontitis as well as patients with poor response to conventional periodontal treatment (SRP). This can be monitored by MMP-8 testing. Despite the lower mean GCF MMP-8 concentrations in smokers, a fraction of smokers sites expressed very high MMP-8 concentrations together with enhanced periodontal activity and could be identified with MMP-8 specific chair-side test. Deep periodontal lesions may be niches for non-periodontopathogenic micro-organisms with systemic effects like C. pneumoniae and possibly play a role in the transmission from one subject to another.

Modern diagnosis of Epstein-Barr virus infections and post-transplant lymphoproliferative disease

Infection by Epstein-Barr virus (EBV) occurs in approximately 95% of the world s population. EBV was the first human virus implicated in oncogenesis. Characteristic for EBV primary infection are detectable IgM and IgG antibodies against viral capsid antigen (VCA). During convalescence the VCA IgM disappears while the VCA IgG persists for life. Reactivations of EBV occur both among immunocompromised and immunocompetent individuals. In serological diagnosis, measurement of avidity of VCA IgG separates primary from secondary infections. However, in serodiagnosis of mononucleosis it is quite common to encounter, paradoxically, VCA IgM together with high-avidity VCA IgG, indicating past immunity. We determined the etiology of this phenomenon and found that, among patients with cytomegalovirus (CMV) primary infection a large proportion (23%) showed antibody profiles of EBV reactivation. In contrast, EBV primary infection did not appear to induce immunoreactivation of CMV. EBV-associated post-transplant lymphoproliferative disease (PTLD) is a life threatening complication of allogeneic stem cell or solid organ transplantation. PTLD may present with a diverse spectrum of clinical symptoms and signs. Due to rapidity of PTLD progression especially after stem cell transplantation, the diagnosis must be obtained quickly. Pending timely detection, the evolution of the fatal disease may be halted by reduction of immunosuppression. A promising new PTLD treatment (also in Finland) is based on anti-CD-20 monoclonal antibodies. Diagnosis of PTLD has been demanding because of immunosuppression, blood transfusions and the latent nature of the virus. We set up in 1999 to our knowledge first in Finland for any microbial pathogen a real-time quantitative PCR (qPCR) for detection of EBV DNA in blood serum/plasma. In addition, we set up an in situ hybridisation assay for EBV RNA in tissue sections. In collaboration with a group of haematologists at Helsinki University Central Hospital we retrospectively determined the incidence of PTLD among 257 allogenic stem cell transplantations (SCT) performed during 1994-1999. Post-mortem analysis revealed 18 cases of PTLD. From a subset of PTLD cases (12/18) and a series of corresponding controls (36), consecutive samples of serum were studied by the new EBV-qPCR. All the PTLD patients were positive for EBV-DNA with progressively rising copy numbers. In most PTLD patients EBV DNA became detectable within 70 days of SCT. Of note, the appearance of EBV DNA preceded the PTLD symptoms (fever, lymphadenopathy, atypical lymphocytes). Among the SCT controls, EBV DNA occurred only sporadically, and the EBV-DNA levels remained relatively low. We concluded that EBV qPCR is a highly sensitive (100%) and specific (96%) new diagnostic approach. We also looked for and found risk factors for the development of PTLD. Together with a liver transplantation group at the Transplantation and Liver Surgery Clinic we wanted to clarify how often and how severely do EBV infections occur after liver transplantation. We studied by the EBV qPCR 1284 plasma samples obtained from 105 adult liver transplant recipients. EBV DNA was detected in 14 patients (13%) during the first 12 months. The peak viral loads of 13 asymptomatic patients were relatively low (<6600/ml), and EBV DNA subsided quickly from circulation. Fatal PTLD was diagnosed in one patient. Finally, we wanted to determine the number and clinical significance of EBV infections of various types occurring among a large, retrospective, nonselected cohort of allogenic SCT recipients. We analysed by EBV qPCR 5479 serum samples of 406 SCT recipients obtained during 1988-1999. EBV DNA was seen in 57 (14%) patients, of whom 22 (5%) showed progressively rising and ultimately high levels of EBV DNA (median 54 million /ml). Among the SCT survivors, EBV DNA was transiently detectable in 19 (5%) asymptomatic patients. Thereby, low-level EBV-DNA positivity in serum occurs relatively often after SCT and may subside without specific treatment. However, high molecular copy numbers (>50 000) are diagnostic for life-threatening EBV infection. We furthermore developed a mathematical algorithm for the prediction of development of life-threatening EBV infection.

New aspects of the diagnosis of Helicobacter pylori infection

Aims: Helicobacter pylori infection, although the prevalence is declining in Western world, is still responsible for several clinically important diseases. None of the diagnostic tests is perfect and in this study, the performance of three stool antigen tests was assessed. In areas of high H. pylori prevalence, the definition of patients with the greatest benefit from eradication therapy may be a problem; the role of duodenal gastric metaplasia in categorizing patients at risk for duodenal ulcer was evaluated in this respect. Whether persistent chronic inflammation and elevated H. pylori antibodies after successful eradication are associated with each other or with atrophic gastritis, a long term sequelae of H. pylori infection, were also studied. Patients and methods: The three stool antigen tests were assessed in pre- and post-eradication settings among 364 subjects in two studies as compared to the rapid urease test (RUT), histology, culture, the 13C-urea breath test (UBT) and enzyme immunoassay (EIA) based H. pylori serology. The association between duodenal gastric metaplasia with duodenal ulcer was evaluated in a retrospective study including 1054 patients gastroscopied due to clinical indications and 154 patients previously operated for duodenal ulcer. The extent of duodenal gastric metaplasia was assessed from histological specimens in different patient groups formed on the basis of gastroscopy findings and H. pylori infection. Chronic gastric inflammation (108 patients) and H. pylori antibodies and serum markers for atrophy (77 patients) were assessed in patients earlier treated for H. pylori. Results: Of the stool antigen tests studied, the monoclonal antibody-based EIA-test showed the highest sensitivity and specificity both in the pre-treatment setting (96.9% and 95.9%) and after therapy (96.9% and 97.8%). The polyclonal stool antigen test and the in-office test had at baseline a sensitivity of 91% and 94%, and a specificity of 96% and 89%, respectively and in a post-treatment setting, a sensitivity of 78% and 91%, and a specificity of 97%, respectively. Duodenal gastric metaplasia was strongly associated with H. pylori positive duodenal ulcer (odds ratio 42). Although common still five years after eradication, persistent chronic gastric inflammation (21%) and elevated H. pylori antibodies (33%) were neither associated with each other nor with atrophic gastritis. Conclusions: Current H. pylori infection can feasibly be diagnosed by a monoclonal antibody-based EIA test with the accuracy comparable to that of reference methods. The performance of the polyclonal test as compared to the monoclonal test was inferior especially in the post-treatment setting. The in-office test had a low specificity for primary diagnosis and hence positive test results should probably be confirmed with another test before eradication therapy is prescribed. The presence of widespread duodenal gastric metaplasia showed promising results in detecting patients who should be treated for H. pylori due to an increased risk of duodenal ulcer. If serology is used later on in patients with earlier successfully treated for H. pylori, it should be taken into account that H. pylori antibodies may persist elevated for years for unknown reason. However, this phenomenon was not found to be associated with persistent chronic inflammation or atrophic changes.

Human trypsinogens in the pancreas and in cancer

Human pancreatic juice contains two major trypsinogen isoenzymes called trypsinogen-1 and -2, or cationic and anionic trypsinogen, respectively. Trypsinogen isoenzymes are also expressed in various normal and malignant tissues. We aimed at developing monoclonal antibodies (MAbs) and time-resolved immunofluorometric methods recognizing human trypsinogen-1 and -2, respectively. Using these MAbs and methods we purified, characterized and quantitated trypsinogen isoenzymes in serum samples, ovarian cyst fluids and conditioned cell culture media. In sera from healthy subjects and patients with extrapancreatic disease the concentration of trypsinogen-1 is higher than that of trypsinogen-2. However, in acute pancreatitis we found that the concentration of serum trypsinogen-2 is 50-fold higher than in controls, whereas the difference in trypsinogen-1 concentration is only 15-fold. This suggested that trypsinogen-2 could be used as a diagnostic marker for acute pancreatitis. In human ovarian cyst fluids tumor-associated trypsinogen-2 (TAT-2) is the predominant isoenzyme. Most notably, in mucinous cyst fluids the levels of TAT-2 were higher in borderline and malignant than in benign cases. The increased levels in association with malignancy suggested that TAT could be involved in ovarian tumor dissemination and breakage of tissue barriers. Serum samples from patients who had undergone pancreatoduodenectomy contained trypsinogen-2. Trypsinogen-1 was detected in only one of nine samples. These results suggested that the expression of trypsinogen is not restricted to the pancreas. Determination of the isoenzyme pattern by ion exchange chromatography revealed isoelectric variants of trypsinogen isoenzymes in serum samples. Intact trypsinogen isoenzymes and tryptic and chymotryptic trypsinogen peptides were purified and characterized by mass spectrometry, Western blot analysis and N-terminal sequencing. The results showed that pancreatic trypsinogen-1 and -2 are sulfated at tyrosine 154 (Tyr154), whereas TAT-2 from a colon carcinoma cell line is not. Tyr154 is located within the primary substrate binding pocket of trypsin, thus Tyr154 sulfation is likely to influence substrate binding. The previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are suggested to be caused by sulfation of Tyr154 in pancreatic trypsinogens.

Prognostic tumour markers in pancreatic cancer

Background. Pancreatic cancer is one of the major causes of cancer death in the industrialised world. The overall survival of patients with ductal pancreatic adenocarcinoma is poor: 5-year survival is only 0.2 to 4%. Tumour stage and histological grade are used as prognostic markers in pancreatic cancer. However, there are differences in survival within stages and histological grades. New, additional and more accurate prognostic tools are needed. Aims. The purpose of this study was to investigate whether the tissue expression of potential and promising tumour markers p27, tenascin C, syndecan-1, COX-2 and MMP-2 are associated with clinicopathological parameters in pancreatic cancer. The expression of p27, tenascin C and syndecan-1 was also evaluated in acute and chronic pancreatitis. The main purpose in the study was to find new prognostic markers for pancreatic adenocarcinoma. Patients. The study included 147 patients with histologically verified pancreatic adenocarcinoma treated at Helsinki University Central Hospital from 1974 to1998. Methods. The expression of tumour marker antigens was demonstrated by immunohistochemistry using monoclonal antibodies against p27, syndecan-1, tenascin C, COX-2 and MMP-2. The results were compared with clinicopathological variables, i.e. age, sex, TNM stage and histological grade. Survival analyses were performed with univariate Kaplan-Meier life-tables and the log-rank test, while multivariate analyses were performed using Cox regression. Results. Pancreatic adenocarcinomas expressed p27, syndecan-1, tenascin C, COX-2 and MMP-2 in 30, 94, 92, 36 and 50% of the samples, respectively. Loss of p27 expression was associated with poor prognosis in stage I and II pancreatic cancer. Stromal syndecan-1 expression was an independent prognostic marker in pancreatic cancer, whereas epithelial syndecan-1 expression predicted better prognosis only in stage I and II disease. Tenascin C expression did not correlate with survival but was associated with differentiation. COX-2 expression was associated with poor outcome and was an independent prognostic factor. Epithelial MMP-2 correlated with poor prognosis in pancreatic cancer. Conclusion: p27 and epithelial syndecan-1 are prognostic markers in early (stage I and II) pancreatic cancer. Stromal syndecan-1, COX-2 and epithelial MMP-2 are prognostic factors in ductal pancreatic adenocarcinoma.

Interrelations between Dry Eye Syndrome and Tear Fluid Phospholipid Transfer Protein

The simplified model of human tear fluid (TF) is a three-layered structure composed of a homogenous gel-like layer of hydrated mucins, an aqueous phase, and a lipid-rich outermost layer found in the tear-air interface. It is assumed that amphiphilic phospholipids are found adjacent to the aqueous-mucin layer and externally to this a layer composed of non-polar lipids face the tear-air interface. The lipid layer prevents evaporation of the TF and protects the eye, but excess accumulation of lipids may lead to drying of the corneal epithelium. Thus the lipid layer must be controlled and maintained by some molecular mechanisms. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. The aim of this thesis was to investigate the presence and molecular mechanisms of lipid transfer proteins in human TF. The purpose was also to study the role of these proteins in the development of dry eye syndrome (DES). The presence of TF PLTP and CETP was studied by western blotting and mass spectrometry. The concentration of these proteins was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. To study the molecular mechanisms involved in PLTP mediated lipid transfer Langmuir monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) was used. Ocular tissue samples were stained with monoclonal antibodies against PLTP to study the secretion route of PLTP. Heparin-Sepharose affinity chromatography was used for PLTP pull-down experiments and co-eluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. To study whether PLTP plays any functional role in TF PLTP-deficient mice were examined. The activity of PLTP was also studied in dry eye patients. PLTP is a component of normal human TF, whereas CETP is not. TF PLTP concentration was about 2-fold higher than that in human plasma. Inactivation of PLTP by heat treatment or immunoinhibition abolished the phospholipid transfer activity in tear fluid. PLTP was found to be secreted from lacrimal glands. PLTP seems to be surface active and is capable of accepting lipid molecules without the presence of lipid-protein complexes. The active movement of radioactively labeled lipids and high activity form of PLTP to acceptor particles suggested a shuttle model of PLTP-mediated lipid transfer. In this model, PLTP physically transports lipids between the donor and acceptor. Protein-protein interaction assays revealed ocular mucins as PLTP interaction partners in TF. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression was demonstrated. Increased tear fluid PLTP activity was observed among human DES patients. These results together suggest a scavenger property of TF PLTP: if the corneal epithelium is contaminated by hydrophobic material, PLTP could remove them and transport them to the superficial layer of the TF or, alternatively, transport them through the naso-lacrimal duct. Thus, PLTP might play an integral role in tear lipid trafficking and in the protection of the corneal epithelium. The increased PLTP activity in human DES patients suggests an ocular surface protective role for this lipid transfer protein.

Mother-Infant Antibodies to Pneumococcal Polysaccharides and Proteins : Transfer and Persistence of Maternal Antibodies and Development of Vaccine-Induced and Naturally Acquired Antibodies in Infants

Symptomless nasopharyngeal carriage of Streptococcus pneumoniae (pneumococcus) is very common in young children. Occasionally the carriage proceeds into mild mucosal diseases, such as sinusitis or acute otitis media, or into serious life-threatening diseases, such as pneumonia, sepsis or meningitis. Each year, up to one million children less than five years of age worldwide die of invasive pneumococcal diseases (IPD). Especially in the low-income countries IPD is a leading health problem in infants; 75% of all IPD cases occur before one year of age. This stresses the need of increased protection against pneumococcus in infancy. Anti-pneumococcal antibodies form an important component in the defence against pneumococcal infection. Maternal immunisation and early infant immunisation are two possible ways by which potentially protective antibody concentrations against pneumococci could be achieved in early infancy. The aim of this thesis is to increase the knowledge of antibody mediated protection against pneumococcal disease in infants and young children. We investigated the transfer of maternal anti-pneumococcal antibodies from Filipino mothers to their infants, the persistence of the transferred antibodies in the infants, the immunogenicity of the 23-valent pneumococcal polysaccharide vaccine (PPV) in infants and the response of the children to a second dose of PPV at three years of age. We also investigated the development of antibodies to pneumococcal protein antigens in relation to culture-confirmed pneumococcal carriage in infants. Serum samples were collected from the mothers, the umbilical cords and from the infants at young age as well as at three years of age. The samples were used to determine the antibody concentrations to pneumococcal serotypes 1, 5, 6B, 14, 18C and 19F, as well as to the pneumococcal proteins PspA, PsaA, Ply, PspC, PhtD, PhtDC and LytC by the enzyme immunoassay. The findings of the present study confirm previously obtained results and add to the global knowledge of responses to PPV in young children. Immunising pregnant women with PPV provides the infants with increased concentrations of pneumococcal polysaccharide antibodies. Of the six serotypes examined, serotypes 1 and 5 were immunogenic already in infants. At three years of age, the children responded well to the second dose of PPV suggesting that maternal and early infant immunisations might not induce hyporesponsiveness to polysaccharide antigens after subsequent immunisations. The anti-protein antibody findings provide useful information for the development of pneumococcal protein vaccines. All six proteins studied were immunogenic in infancy and the development of anti-protein antibodies started early in life in relation to pneumococcal carriage.