Biomedical subjects

Erik Larsson

Publications and source records attributed to Erik Larsson.

At least 19 recordsLinked to original sources

PubMed2006

Lymphocyte propagation from biopsies of kidney allografts.

Morphological evaluation of transplant biopsies, usually using the Banff classification, is the most important tool to diagnose rejection after kidney transplantation. However, morphological analysis only scores the amount and localisation of infiltrating cells, and studies show that up to 30% of grafts with a stable function display infiltration of lymphocytes consistent with acute cellular rejection. Methods to study the functional properties of the infiltrating lymphocytes are therefore needed. We applied a tissue culture system on biopsies from transplanted human kidneys, allowing infiltrating cells to propagate out from the tissue. Cells were then counted and subtyped by flow cytometry. The results were correlated to morphology. In total, 92 biopsies from 69 patients were analysed. For 14 patients, serial biopsies were available. In grafts with cellular or combined cellular and vascular rejection, the number of ex vivo propagated mononuclear cells was higher than from non-rejecting grafts. A similar pattern was seen for CD3(+) T cells as well as for T cells expressing CD25 or MHC class II antigens. However, the proportion of CD25(+) or MHC class II(+) T lymphocytes was similar in all groups (no rejection, vascular rejection, borderline changes, cellular rejection, combined cellular and vascular rejection). In all groups the number of CD4(+) cells was higher than the number of CD8(+) cells. The results confirm previous experimental studies showing that graft-infiltrating cells are possible to culture in vitro and that lymphocyte propagation correlates to acute cellular rejection. Tissue culturing is easy to perform and evaluate and can be used to determine and analyse the cellular immune response to allografts and may thus be used as a complement to morphological analyses.

Antigens, CD↗

PubMed2006

New insights to vascular smooth muscle cell and pericyte differentiation of mouse embryonic stem cells in vitro.

OBJECTIVE: The molecular mechanisms that regulate pericyte differentiation are not well understood, partly because of the lack of well-characterized in vitro systems that model this process. In this article, we develop a mouse embryonic stem (ES) cell-based angiogenesis/vasculogenesis assay and characterize the system for vascular smooth muscle cell (VSMC) and pericyte differentiation. METHODS AND RESULTS: ES cells that were cultured for 5 days on OP9 stroma cells upregulated their transcription of VSMC and pericyte selective genes. Other SMC marker genes were induced at a later time point, which suggests that vascular SMC/pericyte genes are regulated by a separate mechanism. Moreover, sequence analysis failed to identify any conserved CArG elements in the vascular SMC and pericyte gene promoters, which indicates that serum response factor is not involved in their regulation. Gleevec, a tyrosine kinase inhibitor that blocks platelet-derived growth factor (PDGF) spell-receptor signaling, and a neutralizing antibody against transforming growth factor (TGF) beta1, beta2, and beta3 failed to inhibit the induction of vascular SMC/pericyte genes. Finally, ES-derived vascular sprouts recruited cocultured MEF cells to pericyte-typical locations. The recruited cells activated expression of a VSMC- and pericyte-specific reporter gene. CONCLUSIONS: We conclude that OP9 stroma cells induce pericyte differentiation of cocultured mouse ES cells. The induction of pericyte marker genes is temporally separated from the induction of SMC genes and does not require platelet-derived growth factor B or TGFbeta1 signaling.

Animals↗

PubMed2006

Ischemia-induced renal expression of hyaluronan and CD44 in diabetic rats.

BACKGROUND/AIMS: Unilateral renal ischemia during 30 min causes severe, non-reversible renal damage in diabetic (DM) rats, but not in nondiabetic rats. Hyaluronan (HA) is a glycosaminglycan involved in various forms of renal injury. We examined the role of HA and CD44, a major receptor for HA, in the development of postischemic renal injury in DM rats. METHODS: The left renal artery of streptozotocin diabetic Wistar rats was clamped for 30 min. The HA content in the kidneys was measured. A biotinylated HA-binding probe was used to localize HA. Inflammatory cells and other cells expressing CD44 were identified by immunohistochemistry. RESULTS: In ischemic DM kidneys the renal HA-content started to increase already after 24 h and significantly so after 1-8 weeks after ischemia/reperfusion (I/R). The relative water content of the kidneys increased in parallel. HA started to appear in the cortex of ischemic DM kidneys 1 week after I/R. In contrast, the non-DM ischemic kidneys showed no increase of HA and water content after 1-8 weeks after I/R. The tubular cells in the cortex and outer medulla demonstrated increased staining for CD44. In the same compartments the increased numbers of infiltrating inflammatory cells also expressed CD44. CONCLUSION: HA-accumulation in the renal cortex might contribute to the renal damage seen after transient ischemia in DM rats by promoting inflammation through interaction between HA and CD44 expressing inflammatory cells. Furthermore, HA accumulation may contribute to an interstitial renal edema.

Animals↗

PubMed2006

The invasive behaviour of prostatic cancer cells is suppressed by inhibitors of tyrosine kinase.

Proteolytic enzymes, and especially urokinase plasminogen activator (uPA), play an important role in tumour invasion and metastasis. Previously we demonstrated that the production of urokinase plasminogen activator (uPA) was decreased by several tyrosine kinase inhibitors (TKI) in two prostatic carcinoma cell lines. The effect of the two TKI genistein and tyrphostin AG-1478 was investigated in the prostate carcinoma cell lines PC-3 and DU-145. A reconstituted basal lamina (Matrigel) was used as a migration barrier. The production of matrix metalloproteinases (MMP) was also measured. Roles of plasminogen and uPA were examined. Cell invasion was increased by plasminogen, but this enhanced cell migration was counteracted by TKI treatment. The increased cell invasion induced by plasminogen was decreased by at least 60% in both cell lines when alpha-2 anti-plasmin was added to the assay. Cells in the absence of plasminogen were not affected by TKI. External uPA failed to regenerate the decreased cell invasion caused by TKI. The production of MMP was inhibited by both TKI. Our results indicate a possible role of TKI as inhibitors of cancer cell invasion by inhibiting uPA and MMP production.

Basement Membrane↗

PubMed2006

Urokinase plasminogen activator receptor (uPAR) expression is reduced by tyrosine kinase inhibitors.

Previously we reported that tyrosine kinase inhibitors (TKI) produced a reduction in uPA expression in prostatic cancer cells, and that TKI-treated cells were less invasive compared to untreated cells. Nevertheless, no change in cell migration was observed when TKI-treated cells were supplied with external uPA, thus indicating more complex mechanisms leading to decreased cell invasion. uPAR expression was measured with an enzyme-linked immunosorbent assay (ELISA) in PC-3 and DU-145 prostate carcinoma cells treated with the two TKI genistein and AG-1478. uPAR mRNA levels were measured with real-time reverse transcriptase-polymerase chain reaction (RT-PCR). uPAR immunocytochemistry was used to examine the receptor distribution in cells grown on a reconstituted basal lamina. Immunocytochemistry showed an intense uPAR immunostaining in invading cells, particularly in the leading edge membrane. Treatment with genistein and AG-1478 led to a decreased expression of uPAR in DU-145, but not in PC-3. Furthermore, a reduction of uPAR mRNA was found in TKI-treated DU-145 cells, while PC-3 was not affected. Our results indicate a possible role of TKI as cancer suppressors by acting as a regulator of uPAR expression.

Adenocarcinoma↗

PubMed2006

Tyrosine kinase inhibitors alter adhesivity of prostatic cancer cells to extracellular matrix components.

Tyrosine kinase inhibitors (TKIs) are thought to have potential as a new generation of anti-cancer drugs. Since invasiveness, the main characteristic of malignant behaviour, is believed to depend on altered cell-matrix interactions, we investigated the effect of two potent TKIs, genistein and tyrphostin AG-1478, on the interaction of prostate cancer cells with extracellular matrix components. PC-3 and DU-145 cells were treated with various concentrations of genistein and tyrphostin AG-1478. Adhesion to extracellular matrix was assayed using fluorescence-labelled cells seeded on collagen type I, collagen type IV, fibronectin, laminin and vitronectin. The expression levels of integrin beta1, alpha2, alpha3 and alpha5 subunits were measured using flow cytometry of cells labelled with monoclonal murine antibodies. Genistein treatment reduced the ability of both cell lines to adhere to the matrix proteins tested. This effect was more pronounced for PC-3 cells than for DU-145 cells. Genistein treatment decreased the expression of beta1 integrins by 40% in PC-3 cells and 22% in DU-145. AG-1478 treatment slightly reduced the ability of DU-145 cells to adhere, but did not decrease PC-3 cell adhesion. Nevertheless, expression levels were reduced for most integrins tested, except the expression of alpha-5, for which no significant effect was measured. Our results point to a possible role of TKIs as suppressors of prostate carcinoma cell adhesion to extracellular matrix components, by acting as inhibitors of integrin expression.

Cell Adhesion↗

PubMed2006

Transcription levels of invasion-related genes in prostate cancer cells are modified by inhibitors of tyrosine kinase.

Previous reports have shown that genistein and tyrphostin AG-1478, two tyrosine kinase inhibitors (TKIs), exert multiple cellular effects in prostate carcinoma cells, e.g. a reduction in the production of urokinase plasminogen activator (uPA) and its receptor uPAR, and a decrease in the cells' ability to invade an artificial basement membrane. Microarray technology was used to measure alterations in mRNA levels caused by TKI treatment in two prostatic carcinoma cell lines, PC-3 and DU-145. Genistein treatment led to a reduction of at least 50% in 78 genes in PC-3, while 82 were twofold upregulated. In DU-145, the same treatment resulted in a 50% decreased transcript level in 120 genes, and increased expression in 25 genes. Tyrphostin AG-1478 produced a 50% reduction in mRNA levels in 58 genes in DU-145, whereas no alterations were demonstrated using the tyrphostin in PC-3 cells. Among the effects of TKIs, a lowered uPA and uPAR transcription was demonstrated in genistein-treated cells, while a few metalloproteinases (MMPs) were affected. Transcription of various integrin subunits was also downregulated overall. Several alterations in gene transcription were demonstrated in PC-3 and DU-145 after TKI treatment. This knowledge could be of importance in the search for new therapeutic strategies in prostate cancer treatment, and the interplay between the various effects needs to be investigated further.

Antineoplastic Agents↗

PubMed2005

Predictive screening for regulators of conserved functional gene modules (gene batteries) in mammals.

BACKGROUND: The expression of gene batteries, genomic units of functionally linked genes which are activated by similar sets of cis- and trans-acting regulators, has been proposed as a major determinant of cell specialization in metazoans. We developed a predictive procedure to screen the mouse and human genomes and transcriptomes for cases of gene-battery-like regulation. RESULTS: In a screen that covered approximately 40 percent of all annotated protein-coding genes, we identified 21 co-expressed gene clusters with statistically supported sharing of cis-regulatory sequence elements. 66 predicted cases of over-represented transcription factor binding motifs were validated against the literature and fell into three categories: (i) previously described cases of gene battery-like regulation, (ii) previously unreported cases of gene battery-like regulation with some support in a limited number of genes, and (iii) predicted cases that currently lack experimental support. The novel predictions include for example Sox 17 and RFX transcription factor binding sites that were detected in approximately 10% of all testis specific genes, and HNF-1 and 4 binding sites that were detected in approximately 30% of all kidney specific genes respectively. The results are publicly available at http://www.wlab.gu.se/lindahl/genebatteries. CONCLUSION: 21 co-expressed gene clusters were enriched for a total of 66 shared cis-regulatory sequence elements. A majority of these predictions represent novel cases of potential co-regulation of functionally coupled proteins. Critical technical parameters were evaluated, and the results and the methods provide a valuable resource for future experimental design.

Amino Acid Motifs↗

PubMed2005

Craniofacial and dentofacial development in pigs fed soft and hard diets.

INTRODUCTION: Several authors have found a correlation between reduced chewing activity and malocclusion. In animal experiments, it has been possible to correlate a diet of low chewing resistance to narrower arches, which predispose the subject to crowding and irregular teeth. METHODS: In this study, 17 pigs were weaned at 5 weeks of age and divided into 2 groups according to diet and housing. The soft-diet, indoor group was housed in conventional pens, and the animals were fed aliquots of barley and oats with soya and a mineral-vitamin premix added. The food was mixed with water at a dry matter content of about 25%. The hard-diet, outdoor group was kept outdoors; the pigs were fed solid food and also ate organic matter in the soil. All pigs were killed at 22 months of age. The skulls were dissected, and transverse and sagittal craniofacial and dentofacial dimensions were measured. RESULTS: Chewing hard food caused considerable occlusal and approximal attrition in the experimental animals. The dental arches were shorter due to this attrition and to mesial migration of the molars and premolars. The approximal attrition also reduced the tendency for crowding and rotation of the teeth. Posterior crossbite was more common among the hard-chewing animals. CONCLUSIONS: The increase in arch width in the hard-diet animals reported in the literature was not duplicated in this study. On the contrary, the soft-diet pigs had significantly wider arches than the hard-diet animals. This was most pronounced in the premolar region and could be the result of an atypical tongue habit, caused by the nonphysiologic feeding of the pigs. The soft-diet pigs also had a greater tendency to postnormal occlusion, especially in the canine region.

Animals↗

PubMed2005

Dosimetry calculations on a tissue level by using the MCNP4c2 Monte Carlo code.

OBJECTIVE: The aim of this study was to develop a MCNP4c2-code and to further refine the small-scale anatomy intestinal dosimetry model based on a EGS4-code developed by Jonsson et al.(1,2) METHOD: The small intestine was modeled as a hexagonal tube system and includes cross-dose contribution from activity in nearby intestine loops. The model includes villi (height, 500 microm), radiosensitive crypt cells (height, 150 microm), and an overlying mucus layer of thicknesses (5-200 microm). The developed intestinal model used in either of the two Monte Carlo codes make it possible to calculate S-values and subsequent mean absorbed dose to the radiation-sensitive crypt cells in the small intestinal wall by considering contributions from the self-dose and from the cross-dose from nearby intestinal loops. Results are given for monoenergetic electrons and photons and for full decay schemes of (99m)Tc, (111)In, (131)I, (67)Ga, (90)Y, and (211)At. RESULTS: Results show that the cross-dose from nearby intestinal loops is significant, and that the fraction of cumulated activity in the intestinal wall contents is important for accurate absorbed-dose estimation. CONCLUSION: It is evident from our study that previous Medical Internal Radiation Dose (MIRD) and International Conference on Radiological Protection (ICRP) models tend to overestimate the absorbed dose to the wall. Our work on the gastrointestinal tract model includes several noticeable refinements, as compared to the MIRD- and ICRP model, and the "onion shell" geometry can easily be transferred to similar geometrical dosimetry applications.

Astatine↗

PubMed2005

Inhibitors of tyrosine kinase inhibit the production of urokinase plasminogen activator in human prostatic cancer cells.

Urokinase-type plasminogen activator (uPA) seems to be an important protease in prostate cancer invasion, and tyrosine phosphorylation is thought to play a role in the regulation of its production. The amount of uPA was measured with a synthetic peptide substrate after treatment with various concentrations of tyrosine kinase inhibitors (TKI). The effect on proliferation and apoptosis was also assayed. Non-toxic levels of genistein or the tyrphostin AG 490 produced up to 50% reduction of the uPA production in PC-3 and DU-145. The tyrphostins AG 1296 and AG 1478 inhibited uPA production in PC-3 cells, whereas DU-145 showed a slight increase of uPA production. TKI neither induced any detectable apoptosis, nor was there any reduction in proliferation rate. TKI can profoundly modify the production of uPA in prostatic cancer cells, thus indicating their possible use as suppressors of the invasive phenotype. The therapeutic potential of TKI warrants further investigation.

Antineoplastic Agents↗

PubMed2005

ERV3 and related sequences in humans: structure and RNA expression.

The ERV3 locus at chromosome 7q11 is a much studied human endogenous retroviral (HERV) sequence, owing to an env open reading frame (ORF) and placental RNA and protein expression. An analysis of the human genome demonstrated that ERV3 is one of a group of 41 highly related elements (ERV3-like HERVs) which use proline, isoleucine, or arginine tRNA in their primer binding sites. In addition to elements closely related to ERV3, the group included the previously known retinoic acid-inducible element, RRHERVI, also referred to as HERV15, but was separate from the related HERV-E elements. The ERV3-like elements are defective. The only element with an ORF among gag, pro, pol, and env genes was the env ORF of the original ERV3 locus. A search in dbEST revealed ERV3 RNA expression in placenta, skin, carcinoid tumor, and adrenal glands. Expression was also studied with newly developed real-time quantitative PCRs (QPCR) of ERV3 and HERV-E(4-1) env sequences. Results from a novel histone 3.3 RNA QPCR result served as the expression control. QPCR results for ERV3 were compatible with previously published results, with a stronger expression in adrenal gland and placenta than in 15 other human tissues. The expression of the envelope (env) of ERV3 at chromosome 7q11 was also studied by using stringent in situ hybridization. Expression was found in corpus luteum, testis, adrenal gland, Hassal's bodies in thymus, brown fat, pituitary gland, and epithelium of the lung. We conclude that ERV3 env is most strongly expressed in adrenal and sebaceous glands as well as in placenta.

Amino Acid Sequence↗

PubMed2004

Effect of food consistency on temporomandibular joint morphology: an experimental study in pigs.

AIMS: To investigate whether there are any correlations between increased masticatory loading, degree of tooth wear, and the size, form, and macroscopic surface of the temporomandibular joints (TMJs). METHODS: The degree of tooth wear and different TMJ variables were compared in 2 groups of domestic pigs. One group of 8 pigs had been raised indoors (ID group), and the other group of 9 pigs had been raised outdoors (OD group). The pigs in the ID group were fed a soft diet and were not provided any straw in their pens. The OD group was fed a solid diet and could also grub in the soil, resulting in an exposure to more abrasive components and to greater chewing demands. All pigs were sacrificed at the age of 22 months. RESULTS: The pigs in the OD group exhibited significantly more tooth wear compared to the ID group. No difference in mediolateral size of the condyles could be found between the 2 groups. Form and surface changes of the TMJs varied substantially between individuals, but not between the 2 groups. No correlation could be found between the degree of tooth wear and any of the TMJ variables. CONCLUSION: Exposure to a tougher diet containing more abrasive substances has a significant impact on the degree of tooth wear but seems to have no consequences either for the size of the TMJ condyles or for form or surface changes of the TMJs.

Animal Feed↗

PubMed2004

Factors that influence the proclination or retroclination of the lower incisors in children with prolonged thumb-sucking habits.

In some children with a prolonged finger-sucking habit, the lower incisors are retroclined, however, proclination of the lower incisors has been frequently observed. The purpose of this study was to determine how such differences develop in the child with a digit-sucking habit. Children with a prolonged thumb-sucking habit and proclination of the lower incisors (Ili/ML > 97 degrees) were cephalometrically and clinically compared to persistent thumb-suckers with retroclined lower incisors (Ili/ML < 89 degrees). In the group with retroclined lower incisors the angle between the thumb and the lower incisors was significantly smaller and the thickness of the lower lip significantly thinner than in the group with proclined incisors. A higher frequency of early loss of deciduous molars was also observed in the group with retroclined incisors. The tightness of the lower lip, early loss of deciduous molars and the angle between the teeth and the thumb are factors which have been identified to influence the inclination of the lower incisors in persistent digit suckers. The majority of children with prolonged thumb-sucking have proclined lower incisors rather than retroclined lower incisors.

Adolescent↗

PubMed2003

Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins.

Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were co-immunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/ Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase ('MAGUK') family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr(389), located in the alphaB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr(389) to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.

Amino Acid Sequence↗

PubMed2003

Longitudinal changes in gingival condition in crowded and noncrowded dentitions subjected to fixed orthodontic treatment.

A previous study showed that initial crowding in the dentition was negatively correlated with gingival bleeding at the end of orthodontic treatment, whereas visible plaque on the labial surfaces had apparently no significant association with gingival health. The present study further examined the relationship between initial crowding and gingival health during fixed orthodontic treatment. A total of 220 patients were followed longitudinally during treatment. In this population, 2 groups, 1 consisting of 45 patients with severe crowding and 1 consisting of 52 patients with no crowding, were examined more closely. The gingival bleeding index (GBI) was recorded at bonding, at 12, 24, and 48 weeks after bonding, and at debonding. At bonding, the GBI was the same in the 2 groups. However, the GBI improved significantly from 12 weeks after bonding to debonding in the patients with crowded dentitions, whereas the patients with noncrowded dentitions showed no changes. Possible reasons for this difference were identified: to have the same GBI at bonding, despite the severe crowding, the patients in this group probably had better oral hygiene. Those with crowded dentitions had received more oral hygiene instruction within 2 years before bonding than those with noncrowded dentitions, according to the general practitioners' records. When the crowding was eliminated after approximately 3 months, the conditions for cleaning the teeth were improved, and, accordingly, the patients' improved oral hygiene resulted in lowered GBI scores. One could also speculate that correcting the crowding during the first few months of treatment had a positive psychological effect, motivating the patients to use the skills in oral hygiene that they had adopted before treatment.

Adolescent↗

PubMed2002

Rat parietal cells express CCK(2) receptor mRNA: gene expression analysis of single cells isolated by laser-assisted microdissection.

Gastrin plays a crucial role in maintaining a normal cellular composition and function of the oxyntic mucosa. It has been debated for decades whether parietal cells possess cholecystokinin-2 (CCK(2)) receptors and interact directly with gastrin. We investigated whether parietal cells express CCK(2) receptor mRNA by using new molecular biology techniques. Rat oxyntic mucosal cells were dispersed and enriched by elutriation, and single parietal and ECL cells were isolated from cell smears by means of laser microbeam microdissection and laser pressure catapulting. The mRNA from each single cell was isolated and subjected to one-step multiplex or conventional reverse transcription-polymerase chain reaction and subsequent nested PCR. Specific primers for the CCK(2) receptor were used in combination with primers for H,K-ATPase and histidine decarboxylase, specific markers for parietal and ECL cells, respectively. CCK(2) receptor mRNA was detected in 25% of the rat parietal cells and 40% of the ECL cells examined.

Animals↗

PubMed2002

[Sharper diagnostic tool the future promise of laser-assisted microdissection].

The paper describes the use of laser-assisted microdissection to retrieve microscopically defined cell populations including single cells from tissue sections for subsequent analysis of genomic DNA and mRNA. A general background is given on the techniques available and requirements for PCR based on minute templates. Different pre-PCR approaches are briefly described and possibilities and limitations of using archival material compared to fresh frozen tissue are discussed. In the article we give one example on how we have used the PALM laser microscopy system in combination with a nested, multiplex PCR system to analyze single normal keratinocytes as well as tumor cells from a case of basal cell cancer. We found that p53 mutations are common in normal, chronically sun-exposed skin. Widespread yet common mutations in the p53 gene that were unrelated to immunoreactivity for the p53 antibody were found in tumor cells. In addition there were rare mutations in occasional tumor cells that apparently did not result in selective growth advantage. Perspectives for the future are presented and the potential of laser assisted microdissection is highlighted within the fields of cancer research, developmental studies as well as studies of inflammatory and degenerative diseases. The combination of a method that allows careful selection of defined cells with powerful micro array based techniques, provides a setting with potential to uncover pathogenic mechanisms for large variety of human diseases.

Cytological Techniques↗