In vitro development of islets from human adult pancreatic tissues

Transplantation of isolated islets from cadaver pancreas is a promising possibility for the optimal treatment of type 1 diabetes. The lack of islets is a major problem. Here we have investigated the possibility of generating islets in tissue culture of human pancreatic cells. We first reproduced a previously reported method of in vitro generation of endocrine cells from human adult pancreatic tissue. By tracing the bromodeoxyuridine-labeled cells in differentiated islet buds, we found that the pancreatic progenitor cells represented a subpopulation of cytokeratin 19 (CK19)-positive ductal cells. Serum-free medium and Matrigel overlay were essential for the endocrine differentiation. We then examined the involvement of preexisting islet cells in islet neogenesis. About 6-10% of endocrine cells dedifferentiated and acquired a transitional phenotype by coexpressing CK19. Significant cell proliferation was only observed in CK19-positive cells, but not in chromogranin A-positive endocrine cells. The in vitro-derived human islets were morphologically and functionally immature when compared with normal islets. Their insulin mRNA levels were only 4-5% of that found in fresh human islets, and glucose-stimulated insulin release was 3 times lower than that of control islets. Moreover, some immature endocrine cells coexpressed insulin and glucagon. After transplantation in nude mice, the in vitro-generated islets became mature with one type of hormone per endocrine cell. In addition, we also found that also in both fresh islet transplants many cells coexpressed endocrine markers and ductal marker CK19 as a sign of ductal to endocrine cell transition. Finally, we studied the effects of clinically used immunosuppressive drugs on precursor cell proliferation and differentiation. Mycophenolate mofetil (MMF) severely hampered duct-cell proliferation, and significantly reduced the total DNA content indicating its antiproliferative effect on the precursors. Tacrolimus mainly affected differentiated beta cells by decreasing the insulin content per DNA as well as the proportion of insulin-positive cells. Sirolimus and daclizumab did not show any individual or synergistic side effects suggesting that these drugs are amenable for use in clinical islet transplantation. In summary, we confirm the capacity of endocrine differentiation from progenitors present in the adult human pancreas. The plasticity of differentiated cell types of human pancreas may be a potential mechanism of human pancreas regeneration. Ductal cell differentiation into endocrine cells in transplanted islets may be an important factor in sustaining the long-term function of islet transplants. The immunosuppressive protocol is likely to be an important determinant of long-term clinical islet graft function. Moreover, these results provide new information on the mechanisms of pancreatic islet regeneration and provide the basis for the development of new strategies for the treatment of insulin deficient diabetes mellitus.

Nutrition and bone metabolism : In vivo effects of inorganic phosphate on rats and in vitro effects of bioactive tripeptides on human osteoblasts

Nutrition affects bone health throughout life. To optimize peak bone mass development and maintenance, it is important to pay attention to the dietary factors that enhance and impair bone metabolism. In this study, the in vivo effects of inorganic dietary phosphate and the in vitro effects of bioactive tripeptides, IPP, VPP and LKP were investigated. Dietary phosphate intake is increased through the use of convenience foods and soft drinks rich in phosphate-containing food additives. Our results show that increased dietary phosphate intake hinders mineral deposition in cortical bone and diminishes bone mineral density (BMD) in the aged skeleton in a rodent model (Study I). In the growing skeleton (Study II), increased phosphate intake was observed to reduce bone material and structural properties, leading to diminished bone strength. Studies I and II revealed that a low Ca:P ratio has negative effects on the mature and growing rat skeleton even when calcium intake is sufficient. High dietary protein intake is beneficial for bone health. Protein is essential for bone turnover and matrix formation. In addition, hydrolysis of proteins in the gastrointestinal tract produces short peptides that possess a biological function beyond that of being tissue building blocks. The effects of three bioactive tripeptides, IPP, VPP and LKP, were assessed in short- and long-term in vitro experiments. Short-term treatment (24 h) with tripeptide IPP, VPP or LKP influenced osteoblast gene expression (Study III). IPP in particular, regulates genes associated with cell differentiation, cell growth and cell signal transduction. The upregulation of these genes indicates that IPP enhances osteoblast proliferation and differentiation. Long-term treatment with IPP enhanced osteoblast gene expression in favour of bone formation and increased mineralization (Study IV). The in vivo effects of IPP on osteoblast differentiation might differ since eating frequency drives food consumption, and protein degradation products, such as bioactive peptides, are available periodically, not continuously as in this study. To sum up, Studies I and II raise concern about the appropriate amount of dietary phosphate to support bone health as excess is harmful. Studies III and IV in turn, support findings of the beneficial effects of dietary protein on bone and provide a mechanistic explanation since cell proliferation and osteoblast function were improved by treatment with bioactive tripeptide IPP.

Activator Protein-1 and Epidermal Growth Factor Receptor Interplay : In Vivo & In Vitro Studies

Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.

Spectroscopy of tissue triglyceride composition : In vitro and in vivo studies

Lipid analysis is commonly performed by gas chromatography (GC) in laboratory conditions. Spectroscopic techniques, however, are non-destructive and can be implemented noninvasively in vivo. Excess fat (triglycerides) in visceral adipose tissue and liver is known predispose to metabolic abnormalities, collectively known as the metabolic syndrome. Insulin resistance is the likely cause with diets high in saturated fat known to impair insulin sensitivity. Tissue triglyceride composition has been used as marker of dietary intake but it can also be influenced by tissue specific handling of fatty acids. Recent studies have shown that adipocyte insulin sensitivity correlates positively with their saturated fat content, contradicting the common view of dietary effects. A better understanding of factors affecting tissue triglyceride composition is needed to provide further insights into tissue function in lipid metabolism. In this thesis two spectroscopic techniques were developed for in vitro and in vivo analysis of tissue triglyceride composition. In vitro studies (Study I) used infrared spectroscopy (FTIR), a fast and cost effective analytical technique well suited for multivariate analysis. Infrared spectra are characterized by peak overlap leading to poorly resolved absorbances and limited analytical performance. In vivo studies (Studies II, III and IV) used proton magnetic resonance spectroscopy (1H-MRS), an established non-invasive clinical method for measuring metabolites in vivo. 1H-MRS has been limited in its ability to analyze triglyceride composition due to poorly resolved resonances. Using an attenuated total reflection accessory, we were able to obtain pure triglyceride infrared spectra from adipose tissue biopsies. Using multivariate curve resolution (MCR), we were able to resolve the overlapping double bond absorbances of monounsaturated fat and polyunsaturated fat. MCR also resolved the isolated trans double bond and conjugated linoleic acids from an overlapping background absorbance. Using oil phantoms to study the effects of different fatty acid compositions on the echo time behaviour of triglycerides, it was concluded that the use of long echo times improved peak separation with T2 weighting having a negligible impact. It was also discovered that the echo time behaviour of the methyl resonance of omega-3 fats differed from other fats due to characteristic J-coupling. This novel insight could be used to detect omega-3 fats in human adipose tissue in vivo at very long echo times (TE = 470 and 540 ms). A comparison of 1H-MRS of adipose tissue in vivo and GC of adipose tissue biopsies in humans showed that long TE spectra resulted in improved peak fitting and better correlations with GC data. The study also showed that calculation of fatty acid fractions from 1H-MRS data is unreliable and should not be used. Omega-3 fatty acid content derived from long TE in vivo spectra (TE = 540 ms) correlated with total omega-3 fatty acid concentration measured by GC. The long TE protocol used for adipose tissue studies was subsequently extended to the analysis of liver fat composition. Respiratory triggering and long TE resulted in spectra with the olefinic and tissue water resonances resolved. Conversion of the derived unsaturation to double bond content per fatty acid showed that the results were in accordance with previously published gas chromatography data on liver fat composition. In patients with metabolic syndrome, liver fat was found to be more saturated than subcutaneous or visceral adipose tissue. The higher saturation observed in liver fat may be a result of a higher rate of de-novo-lipogenesis in liver than in adipose tissue. This thesis has introduced the first non-invasive method for determining adipose tissue omega-3 fatty acid content in humans in vivo. The methods introduced here have also shown that liver fat is more saturated than adipose tissue fat.

Inhibition of CYP1A2-mediated drug metabolism in vitro and in humans : With special emphasis on rofecoxib and other NSAIDs

The cytochrome P450 1A2 (CYP1A2) is one of the major metabolizing enzymes. The muscle relaxant tizanidine is a selective substrate of CYP1A2, and the non-steroidal anti-inflammatory drug (NSAID) rofecoxib was thought to modestly in-hibit it. Cases suggesting an interaction between tizanidine and rofecoxib had been reported, but the mechanism was unknown. Also other NSAIDs are often used in combination with muscle relaxants. The aims of this study were to investigate the effect of rofecoxib, several other NSAIDs and female sex steroids on CYP1A2 ac-tivity in vitro and in vivo, and to evaluate the predictability of in vivo inhibition based on in vitro data. In vitro, the effect of several NSAIDs, female sex steroids and model inhibitors on CYP1A2 activity was studied in human liver microsomes, without and with preincubation. In placebo controlled, cross-over studies healthy volunteers ingested a single dose of tizanidine after a pretreament with the inhibitor (rofecoxib, tolfenamic acid or celecoxib) or placebo. Plasma (and urine) concentrations of tizanidine and its metabolites were measured, and the pharmacodynamic effects were recorded. A caffeine test was also performed. In vitro, fluvoxamine, tolfenamic acid, mefenamic acid and rofecoxib potently in-hibited CYP1A2. Ethinylestradiol, celecoxib, desogestrel and zolmitriptan were moderate, and etodolac, ciprofloxacin, etoricoxib and gestodene were weak inhibi-tors of CYP1A2. At 100 µM, other tested NSAIDs and steroids inhibited CYP1A2 less than 35%. Rofecoxib was found to be a mechanism-based inhibitor of CYP1A2. In vivo, rofecoxib greatly increased the plasma concentrations (over ten-fold) and the pharmacodynamic effects of tizanidine. Also the metabolism of caf-feine was impaired by rofecoxib. Despite the relatively strong in vitro CYP1A2 inhibitory effects, tolfenamic acid and celecoxib did not have a significant effect on tizanidine and caffeine concentrations in humans. Competitive inhibition model and the free plasma concentration of the inhibitor predicted well the effect of fluvoxam-ine and the lack of effect of tolfenamic acid and celecoxib on tizanidine concentra-tions in humans, and mechanism-based inhibition model explained the effects of rofecoxib. However, the effects of ciprofloxacin and oral contraceptives were un-derestimated from the in vitro data. Rofecoxib is a potent mechanism-based inhibitor of CYP1A2 in vitro and in vivo. This mechanism may be involved in the adverse cardiovascular effects of rofecoxib. Tolfenamic acid and celecoxib seem to be safe in combination with tizanidine, but mefenamic acid might have some effect on tizanidine concentrations in vivo. Con-sidering the mechanism of inhibition, and using the free plasma concentration of the inhibitor, many but not all CYP1A2 interactions can be predicted from in vitro data.

X-ray scattering and diffraction enhanced imaging studies of in vitro breast tissues

This thesis is a study of the x-ray scattering properties of tissues and tumours of the breast. Clinical radiography is based on the absorption of the x-rays when passing right through the human body and gives information about the densities of the tissues. Besides being absorbed, x-rays may change their direction within the tissues due to elastic scattering or even to refraction. The phenomenon of scattering is a nuisance to radiography in general, and to mammography in particular, because it reduces the quality of the images. However, scattered x-rays bear very useful information about the structure of the tissues at the supra-molecular level. Some pathologies, like breast cancer, produce alterations to the structures of the tissues, being especially evident in collagen-rich tissues. On the other hand, the change of direction due to refraction of the x-rays on the tissue boundaries can be mapped. The diffraction enhanced imaging (DEI) technique uses a perfect crystal to convert the angular deviations of the x-rays into intensity variations, which can be recorded as images. This technique is of especial interest in the cases were the densities of the tissues are very similar (like in mammography) and the absorption images do not offer enough contrast. This thesis explores the structural differences existing in healthy and pathological collagen in breast tissue samples by the small-angle x-ray scattering (SAXS) technique and compares these differences with the morphological information found in the DEI images and the histo-pathology of the same samples. Several breast tissue samples were studied by SAXS technique in the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Scattering patterns of the different tissues of the breast were acquired and compared with the histology of the samples. The scattering signals from adipose tissue (fat), connective tissue (collagen) and necrotic tissue were identified. Moreover, a clear distinction could be done between the scattering signals from healthy collagen and from collagen from an invasive tumour. Scattering from collagen is very characteristic. It includes several scattering peaks and scattering features that carry information about the size and the spacing of the collagen fibrils in the tissues. It was found that the collagen fibrils in invaded tumours were thinner and had a d-spacing length 0,7% longer that fibrils from healthy tumours. The scattering signals from the breast tissues were compared with the histology by building colour-coded maps across the samples. They were also imaged with the DEI technique. There was a total agreement between the scattering maps, the morphological features seen in the images and the information of the histo- pathological examination. The thesis demonstrates that the x-ray scattering signal can be used to characterize tissues and that it carries important information about the pathological state of the breast tissues, thus showing the potential of the SAXS technique as a possible diagnostic tool for breast cancer.

Spectroscopy of tissue triglyceride composition : In vitro and in vivo studies

Lipid analysis is commonly performed by gas chromatography (GC) in laboratory conditions. Spectroscopic techniques, however, are non-destructive and can be implemented noninvasively in vivo. Excess fat (triglycerides) in visceral adipose tissue and liver is known predispose to metabolic abnormalities, collectively known as the metabolic syndrome. Insulin resistance is the likely cause with diets high in saturated fat known to impair insulin sensitivity. Tissue triglyceride composition has been used as marker of dietary intake but it can also be influenced by tissue specific handling of fatty acids. Recent studies have shown that adipocyte insulin sensitivity correlates positively with their saturated fat content, contradicting the common view of dietary effects. A better understanding of factors affecting tissue triglyceride composition is needed to provide further insights into tissue function in lipid metabolism. In this thesis two spectroscopic techniques were developed for in vitro and in vivo analysis of tissue triglyceride composition. In vitro studies (Study I) used infrared spectroscopy (FTIR), a fast and cost effective analytical technique well suited for multivariate analysis. Infrared spectra are characterized by peak overlap leading to poorly resolved absorbances and limited analytical performance. In vivo studies (Studies II, III and IV) used proton magnetic resonance spectroscopy (1H-MRS), an established non-invasive clinical method for measuring metabolites in vivo. 1H-MRS has been limited in its ability to analyze triglyceride composition due to poorly resolved resonances. Using an attenuated total reflection accessory, we were able to obtain pure triglyceride infrared spectra from adipose tissue biopsies. Using multivariate curve resolution (MCR), we were able to resolve the overlapping double bond absorbances of monounsaturated fat and polyunsaturated fat. MCR also resolved the isolated trans double bond and conjugated linoleic acids from an overlapping background absorbance. Using oil phantoms to study the effects of different fatty acid compositions on the echo time behaviour of triglycerides, it was concluded that the use of long echo times improved peak separation with T2 weighting having a negligible impact. It was also discovered that the echo time behaviour of the methyl resonance of omega-3 fats differed from other fats due to characteristic J-coupling. This novel insight could be used to detect omega-3 fats in human adipose tissue in vivo at very long echo times (TE = 470 and 540 ms). A comparison of 1H-MRS of adipose tissue in vivo and GC of adipose tissue biopsies in humans showed that long TE spectra resulted in improved peak fitting and better correlations with GC data. The study also showed that calculation of fatty acid fractions from 1H-MRS data is unreliable and should not be used. Omega-3 fatty acid content derived from long TE in vivo spectra (TE = 540 ms) correlated with total omega-3 fatty acid concentration measured by GC. The long TE protocol used for adipose tissue studies was subsequently extended to the analysis of liver fat composition. Respiratory triggering and long TE resulted in spectra with the olefinic and tissue water resonances resolved. Conversion of the derived unsaturation to double bond content per fatty acid showed that the results were in accordance with previously published gas chromatography data on liver fat composition. In patients with metabolic syndrome, liver fat was found to be more saturated than subcutaneous or visceral adipose tissue. The higher saturation observed in liver fat may be a result of a higher rate of de-novo-lipogenesis in liver than in adipose tissue. This thesis has introduced the first non-invasive method for determining adipose tissue omega-3 fatty acid content in humans in vivo. The methods introduced here have also shown that liver fat is more saturated than adipose tissue fat.

POP-inhibiittorin KYP-2047 vaikutukset angiogeneesiin in vitro

Angiogeneesi on tärkeä ilmiö elimistön fysiologiassa, mutta myös lukuisissa patologisissa tiloissa. Angiogeneesi on monivaiheinen prosessi, joka sisältää angiogeneesiä indusoivia ja sitä inhiboivia tekijöitä tasapainossa keskenään. Useat tutkimukset puoltavat sitä, että tymosiini ȕ4 (Tȕ4) ja tetrapeptidi Ac-SDKP (N-asetyyliseryyli- aspartyyli-lysyyli-proliini) indusoivat angiogeneesiä in vitro ja in vivo. Tutkimukset viittaavat myös siihen, että prolyylioligopeptidaasi (POP) hydrolysoi peptidifragmentin Ac- SDKP Tȕ4:n (43 ah) proliinin jälkeen. POP on laajalti esiintyvä seriiniproteaasi, joka pystyy pilkkomaan vain alle 30 aminohapon oligopeptidejä. Tȕ4:n tulee siksi pilkkoutua ensin jonkin, vielä tuntemattoman peptidaasin johdosta. POP:ia on löydetty eniten aivoista, minkä vuoksi sitä on tutkittu varsinkin muistin ja oppimisen häiriötiloissa sekä neurodegeneratiivisten sairausten yhteydessä. POP:in todellinen fysiologinen merkitys on kuitenkin vielä selvittämättä. Tämän pro gradun kirjallisuusosiossa selvitetään angiogeneesiin liittyvien tekijöiden yhteyksiä sekä kuvataan angiogeenisten Tȕ4:n, Ac-SDKP:n ja POP:in ominaisuuksia, esiintymistä ja toimintaa. Kokeellisen osion tarkoituksena oli osoittaa, osallistuvatko POP ja Tȕ4 tetrapeptidin Ac-SDKP muodostumiseen ja kapillaarimuodostumiseen ja edelleen, voidaanko POPaktiivisuutta, tetrapeptidi- ja kapillaarimuodostumista estää spesifisellä POP-inhibiittorilla, KYP-2047:llä. Kokeellinen osa oli kaksiosainen. Ensimmäisessä osassa tutkittiin POPaktiivisuutta ja suoritettiin Ac-SDKP –pitoisuusmittauksia ajanjaksolla 0-180 min Wistarkannan rotista tehdyillä homogenaateilla. Tutkimusryhminä olivat 0,1 ja 0,5 μM KYP-2047 (+2 μM Tȕ4), 1:20 (0,625 μM) humaaniperäinen rekombinantti-POP (+ 2 μM Tȕ4), 2 μM Tȕ4 (pos. kontrolli) ja raakahomogenaatti (neg. kontrolli). Toisessa osassa tutkittiin kapillaarimuodostumista ajanjaksolla 0-180 min humaaniperäisillä napanuoralaskimon primaariendoteelisoluilla MatrigelTM Matrix -päällystetyllä 48- kuoppalevyllä, jolle oli siirrostettu 50 000 solua/kuoppa. Naudan seerumilla ja antibiooteilla käsitellyt tutkimusryhmät olivat 5 ja 10 μM KYP-2047 (+4 μM Tȕ4), 1:20 (0,625 μM) humaaniperäinen rekombinantti-POP (+4 μM Tȕ4), 4 μM Tȕ4 (pos. kontrolli) ja DMEM (neg. kontrolli). Kuoppia inkuboitiin ja kapillaarimuodostuminen kuvattiin valomikroskoopilla digitaalikameralla. Kutakin tutkimusryhmää pipetoitiin kolmeen rinnakkaiseen kuoppaan ja kokeet toistettiin neljästi. Sulkeutuneiden kapillaarien lukumäärä laskettiin manuaalisesti ja tuloksista tehtiin tilastollinen analyysi. 7ȕ4:n ja POP:in havaittiin molempien osallistuvan tetrapeptidin AC-SDKP muodostumiseen munuaishomogenaateissa. Primaariendoteelisolut muodostivat selkeitä kapillaareja Matrigelilla, erityisesti POP- ja Tȕ4–ryhmissä. KYP-2047 inhiboi tehokkaasti POP:ia kaikissa kokeissa osoittautuen hyväksi antiangiogeeniseksi yhdisteeksi. Angiogeneesin mekanismien ja POP:in, Tȕ4:n ja Ac-SDKP:n yhteyksien selvittäminen vaatii luonnollisesti vielä lisätutkimuksia.

Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.