Geimsa Staining Research Articles

Diagnosis of Lophomonas blattarum in bronchoalveolar lavage samples

Lophomonas blattarum is a multi-flagellate protozoan which causes bronchopulmonary infection in human. As the culture and molecular diagnosis of Lophomonas blattarum have not yet been developed, direct slide examination from Bronchoalveolar lavage (BAL) or sputum is the best method for diagnosis of Lophomonas with morphological features. In the present study, in order to achieve quick and easy identification of Lophomonas, the sensitivity of different staining techniques has been investigated in comparison with the direct wet slide as the gold standard. Giemsa, Trichrome, and Papanicolaou stained-slides have been examined in patients who had lophomoniasis. Material &Methods BAL samples of patients suspected to lophomoniasis were collected. After confirmation of Lophomonas blattarum by observation in the direct test, the slides were prepared using Geimsa, Trichrome, and Papanicolaou staining for each patient. Results Among 158 BAL specimens sent to Imam Reza hospital lab, 50 samples were positive by direct microscopic examination which was stained by Giemsa, Trichrome, and Papanicolaou techniques. The highest sensitivity was seen for Papanicolaou staining with 16% and then Geimsa and Trichrome staining with 12% and 8%, respectively. Conclusion The results of this study show the Papanicolaou staining method had the best sensitivity compared to Giemsa, Trichrome stained-slides for differential diagnosis of this protozoan from epithelial cells.

Various findings in upper gastrointestinal endoscopy in patients presenting with dyspepsia: A clinical study

Background: Incidence of Dysphagia become more prevalent because of the faulty food habits among the people in recent days.Aim: To study incidence of various oesophageal and gastric diseases with respect to age, sex, occupation, socioeconomic status and other factors in our hospital. To find the upper gastrointestinal morphological features among patients presenting with dyspepsia using endoscopy. To find the prevalence of of H-pylori infection in patients presenting with dyspepsia and screening for oesophago-gastric carcinoma among patients presenting with dyspepsia. Materials and Methods: It is a prospective study conducted on patients who presented with dyspepsia in department of general surgery, sree mookabika institute of medical sciences during the period of September 2020 – January 2021.All the patients were evaluated on basis of chief complaints, clinical examination and investigation and They underwent endoscopy and findings were documented, invasive tests like biopsy, rapid urease test and geimsa staining were done findings were tabulated and analysed . Appropriate statistical methods were applied to provide the below mentioned results.Results: Highest prevalence of dyspepsia belong to the age group of 20-40years. Most common presenting complaint was heart burn followed by nausea/vomiting /epigastric discomfort. Dyspepsia were noted more common in male gender comparing to female gender. Most common endoscopic finding among the study population were gastritis followed by esophagitis along with gastritis. H-pylori infection was present in 42.5% of study population. Malignancy was diagnosed in 2.5% patients with dyspepsia. Clinically significant endoscopic findings were observed in 80.8% of patients with non-investigated dyspepsia. Conclusion: Clinically significant endoscopic findings were observed in 80.8% of patients with non-investigated dyspepsia. Prevalence of large number of inflammatory lesions as a result of increased acid production, a remarkable prevalence of H-Pylori infection and low incidence of malignancy in the study group suggests that the non-investigated patients with dyspepsia may be initially managed medically with acid suppressive therapy or H-pylori eradication in cases suspected to be infected. Endoscopy may be undertaken in patients with recurrent symptoms or in whom drug therapy fails.

Clinical, parasitological and molecular profiles of Cutaneous Leishmaniasis and its associated factors among clinically suspected patients attending Borumeda Hospital, North-East Ethiopia.

BackgroundCutaneous leishmaniasis is one of the most neglected tropical diseases increasing in its public health importance. In Ethiopia over 28 million people are living at risk of infection.MethodInstitution based cross-sectional study was conducted at Borumeda Hospital from February to May 2019. A total 205 leishmaniasis suspected patients were included by systematic random sampling technique. Socio demographic characteristics were collected using pre-tested questionnaires. Parasitological investigation was done from skin slit sample by using Geimsa staining method. Species identification was done by PCR-RFLP. Data were entered in to EpiData version 3.1 and analyzed using SPSS version 20 software. P-value of ≤ 0.05 was considered as statistically significant.ResultA total of 205 participants consisting 59% male and 41% female included in this study. The mean age (±SD) of the study participants was 31.9 (±14.29). The overall prevalence of cutaneous leishmaniasis was 22.4% (46/205). The prevalence in males (13.7%) was higher than in females (8.8%). It was more prevalent in the age group 16-45years old (15.6%). Clinically, 60% of patients’ hade single lesion with 1.55 average number of lesions. About 30.7% of patients’ had indurated plaque type of lesion. Most of the lesions were found on head and face (59%). House near to farmland, presence of hyrax in the village and presence of other cutaneous leishmaniasis cases in the neighborhood were independent predicator of cutaneous leishmaniasis prevalence. L.aethopica was found to be the etiologic agent of cutaneous leishmaniasis in the study participants.ConclusionThe prevalence of cutaneous leishmaniasis was 22.4%, this alerts the need of intervention. It is statistically associated with house near to farm land, presence of other cutaneous leishmaniasis cases in the neighborhood and presence of hyrax in village. Head and face were the most common sites of lesion.

A trial study on a mixture of Acridine and Giemsa stains and modified Quantitative Buffy Coat for detection of malaria parasites

Microscopy after Geimsa staining remains the gold standard technique for detection of malaria parasites while Acridine is a common fluorescent stain that enhances the visibility of parasites. Combination of both stains could further enhance the performance of microscopy technique. The aim of this study was to investigate the efficacy of staining with mixture of Acridine and Giemsa and modified Quantitative Buffy Coat technique in detection of plasmodium parasites. Five hundred and seventy (570) volunteered students attending the Kwara State University Health Center Malete with history malaria fever and tested positive for malaria parasite were re-examined with the two methods being investigated. Two millilitres of the blood sample from each subject was collected into EDTA container and mixed properly. Thick smears were made in duplicate from each sample and properly labeled, stained by Giemsa and Acridine-Giemsa techniques and examined under x100 objective of the microscope while the remaining blood samples were also tested by Modified QBC. The study assessed the Sensitivity, Specificity, Positive Predictive Value and Negative Predictive Value of both techniques with respect to Giemsa microscopy as a gold standard. Findings from the investigation showed that modified QBC and Acredine-Giemsa techniques recorded sensitivity of 100% and 99.7% respectively. Concentrated Acridine-Giemsa stain recorded higher specificity (90.7%) than modified QBC with 82.5% using Geimsa staining as a gold standard. The Positive Predictive Value (PPV) for Modified QBC and Acredine-Giemsa techniques were 96.8% and 96.5% respectively whereas the Negative Predictive Value (NPV) for both methods were 100% and 98.7% respectively. In conclusion, the finding depicts a keen competition between Acridine-Giemsa staining method and Modified QBC technique for detection of malaria parasites and there was no significant difference in the efficacy of both methods with respect to detection of malaria parasite.© 2019 International Formulae Group. All rights reservedKeywords: Acridine-Giemsa, Modified QBC, Efficacy, Microscopy, Gold standard

HELICOBACTER PYLORI

Objectives: To determine the frequency of helicobacter pylori in gallbladder in patient with gall stones disease. Study Design: Cross-sectional study. Place and Duration of Study: This study was conducted at Surgical Unit-I Civil Hospital Karachi, March 2013 to August 2013. Methodology: A total of 281 patients diagnosed to have gall stone on basis of clinical history of pain in right upper abdomen with fatty intolerance confirmed on ultrasoundwho were operated rather by laparoscopic procedure or open surgery were included in the study. Immediately following gallbladder removal, the specimen was collected in a sterile cup. The specimen was sent to Laboratory for presence of H. Pylori using GEIMSA staining and for routine histopathology. Data was collected by a resident in predesigned proforma. Results: A total of 281 Patients diagnosed to have gall stone. Operated most of the cases were 21 to 50 years of age with mean age was 38.41±9.86 years. Out of 281 cases, 170 (60.5%) were female and 111 (39.5%) male. Seventy eight patients (27.76%) was observed obsess (BMI > 30kg/m2). Similarly duration of illness of 58.4% was below and equal to 6 months and 41.6% cases above six months. Frequency of helicobacter pylori in gallbladder of patients with gall stones disease was positive in 72(25.62%) cases. Conclusion: We conclude that H. pylori are also found in the biliary system, suggesting that these bacteria are of etiological importance in gallstoneformation. Early detection of helicobacter pylori and its eradication can prevent development of gall stones.

In vitro evaluation of ruthenium complexes for photodynamic therapy

BackgroundPhotodynamic therapy (PDT) is a promising anti-tumor treatment strategy. Photosensitizer is one of the most important components of PDT. In this work, the anticancer activities of PDT mediated by six new ruthenium porphyrin complexes were screened. The mechanisms of the most efficacious candidate were investigated. MethodsPhotocytotoxicity of the six porphyrins was tested. The most promising complex, Rup-03, was further investigated using Geimsa staining, which indirectly detects reactive oxygen species (ROS) and subcellular localization. Mitochondrial membrane potential (MMP), cell apoptosis, DNA fragmentation, c-Myc gene expression, and telomerase activities were also assayed. ResultsRup-03 and Rup-04 had the lowest IC50 values. Rup-03 had an IC50 value of 29.5±2.3μM in HepG2 cells and 59.0±6.1μM in RAW264.7 cells, while Rup-04 had an IC50 value of 40.0±3.8μM in SGC-7901 cells. The complexes also induced cellular morphological changes and impaired cellular ability to scavenge ROS, and accumulated preferentially in mitochondria and endoplasmic reticulum. Rup-03 reduced MMP levels, induced apoptosis, and repressed both c-Myc mRNA expression and telomerase activity in HepG2 cells. ConclusionsAmong six candidates, Rup-03-mediated PDT is most effective against HepG2 and RAW264.7, with a similar efficacy as that of Rup-04-mediated PDT against SGC-7901 cells. Repression of ROS scavenging activities and c-Myc expression, which mediated DNA damage-induced cell apoptosis and repression of telomerase activity, respectively, were found to be involved in the anticancer mechanisms of Rup-03.

Culture and Differentiation of Monocyte Derived Macrophages Using Human Serum: An Optimized Method

Background: Monocyte derived macrophages (MDMs) are appropriate in vitro models to study the function of macrophages in immune related diseases. Not only most of the methods in literature for efficient MDM culture and differentiation are expensive but also they require specific equipment. However, there are some reports indicating that monocyte enrichment is possible through attachment. Purification and differentiation of macrophages occurs in media containing human serum or platelet depleted plasma without extra supplementations. Different variables such as incubation time and serum concentration affect this simple MDM preparation method. Objectives: Here we represent an optimized simple method for MDM preparation from peripheral blood mononuclear cells (PBMC). Materials and Methods: To introduce an optimized method we accomplished the present descriptive study. After PBMC isolation and monocyte enrichment in complete RPMI 1640 growth media, macrophages were cultured and differentiated using human serum. Efficient phagocytosis was evaluated using heat-inactivated Escherichia coli followed by SYBR staining. Geimsa staining of macrophages was accomplished to visualize the typical morphology under light microscopy. Results: The derived macrophages have the typical morphology of differentiated macrophages and are able to phagocyte the heat inactivated SYBR stained E. coli. Conclusions: This optimized method is a simple and cost effective method to prepare MDM with typical morphology representations able to phagocytosis efficiently.

Helicobacter Pylori Infection in Patients with Chronic Calcular Cholecystitis a Cross- Sectional Study

Aims: Helicobacter pylori was found in the bile and gallbladder, so we are aiming to examine the relationship between H. pylori-like bacteria and chronic calcular cholecystitis. METHODS: This cross-sectional study was conducted at our hospitals from May 2011 to January 2012. One hundred and forty patients with chronic calcular cholecystitis were enrolled in this study. Gastroscopy with gastric or duodenal H.pylori urease test and detection of H. pylori in serum were done before the surgical procedure for all patients. After cholecystectomy, the gallbladder tissue was investigated for H. pylori by rapid urease test and Geimsa stain. RESULTS: Of all 140 patients 62.1 % were females, mean age of our patients was 39.04 ± 7.3 years. H. pylori IgG antibody titre was positive in 32 patients with biliary duodenitis, in 43 patients with biliary gastritis, and in 18 patients with duodenal ulcer. The gastric rapid urease test was positive in 40.7 %. The gallbladder rapid urease test was positive in 25 %. In 24 patients both gastric and gallbladder mucosa tested positive for rapid urease test. In 28 patients, the gastric urease test was positive while the gallbladder mucosa tested negative. In 13 patients, the gallbladder mucosa tested positive while the gastric mucosa was negative. There was a significant correlation between the rapid urease test in the gallbladder and in the gastric or duodenal mucosa (P = 0.002). Modified Geimsa staining of gallbladder specimens revealed that H. pylori-like bacteria were detected in 63.6 %. There was a significant correlation between histopathological examination of gallbladder mucosa stained with Geimsa stain and the gallbladder rapid urease test (P < 0.001), but not correlated with the gastric urease test (P = 0.210). CONCLUSION: Our study demonstrates that H. pylori is present in the gallbladder mucosa in patients with chronic calcular cholecystitis and might be an etiological cause of chronic inflammation`.

PREVALENCE RATE OF CHLAMYDIA TRACHOMATIS INFECTION IN EGYPTIAN INFERTILE WOMEN

Chlamydia trachomatis is one of the most common curable bacterial sexually transmitted infections (STIs) worldwide, and its complications in infected women in terms of pelvic inflammatory disease (PID), infertility and ectopic pregnancy among others with this infection highlights the need of its diagnosis in women with a history of infertility and recurrent spontaneous abortion for their better management.the present study evaluates the relationship between either past or current C. trachomatis infection in asymptomatic Egyptian females with unexplained infertility. This may be needed to be highlighted to attract the attention of clinical investigators. In addition the study compares the effectiveness of the point of care (POC) chlamydia test with polymerase chain reaction (PCR) as a potential tool for rapid Chlamydia diagnosis and screening .In the present study we enrolled women attending the gynecology outpatient department (OPD) of Ain shams educational hospital in the period from January 2013 to September 2013.The case group comprised of seventy five married infertile women. control group of twenty patients who were attending the clinic for other gynecologic purposes The clinician team did a routine gynecological per speculum examination to record signs of infection and collected two cervical swabs and blood samples, The first swab was shaken on a vortex mixerThe contents of the tube were used for direct Geimsa staining and point of care testing. The second swab was shaken on a vortex mixer then it was removed after pressing against the tube wall. And the content was preserved at -70oC until used for DNA extraction and PCR Chlamydia rapid test (CRT) wasperformed,The detection of IgG antibodies against C. trachomatis was performed by using a commercial ELISA kit . Seventy five infertile women and twenty control females were tested for current C,trachomatis infection by PCR, C.trachomatis POC test and geimsa staining. AS well as serum samples were collected to detect C.trachomatis specific IgG by ELISA. The Participants of the infertility cases were between 20-45 years old with a mean of (34.18± 5.3) The overall prevalence of anti C trachomatis antibodies (ACTA) IgG in infertile women was 27/75 (36%) in infertile women and 2/20 (10%) in control group ,the difference was statistically significant (PC.trachomatis by PCR was (22/75)29.3% in infertile women and 1/20 (5%) in control subjects versus (18/75) 24% by C.trachomatis POCT. Sensitivity, specificity, Positive predictive value and Negative predictive value of C.trachomatis POCT was compared to PCR for the diagnosis of C.trachomatis infection and it revealed 81.8%, 98.1%, 94.7% and 92.9% respectivelyconclusion, we find a strong relationship between past and current C.trachomatis infection and infertility in a sample of Egyptian infertile women. No difference was found in the prevalence of current or past C.trachomatis infection andfertile controls. The Chlamydia rapid test achieves relatively high diagnostic sensitivity and provides results within 30 minutes. It is suitable as primary diagnostic tool for Chlamydia infection and in settings where PCR tests was limited or absent thus it could be used as a screening tool.

Adolescent pregnancy and the risk of Plasmodium falciparum malaria and anaemia—A pilot study from Sekondi-Takoradi metropolis, Ghana

The problem of malaria in adolescence has been surpassed by the immense burden of malaria in children, most especially less than 5. A substantial amount of work done on malaria in pregnancy in endemic regions has not properly considered the adolescence. The present study therefore aimed at evaluating the prevalence of Plasmodium falciparum and anaemia infection in adolescent pregnant girls in the Sekondi-Takoradi metropolis, Ghana. The study was carried out at four hospitals in the Sekondi-Takoradi metropolis of the western region of Ghana from January 2010 to October 2010. Structured questionnaires were administered to the consenting pregnant women during their antenatal care visits. Information on education, age, gravidae, occupation and socio-demographic characteristics were recorded. Venous bloods were screened for malaria using RAPID response antibody kit and Geimsa staining while haemoglobin estimations were done by cyanmethemoglobin method. The results revealed that adolescent pregnant girls were more likely to have malaria infection than the adult pregnant women (34.6% verses 21.3%, adjusted OR 1.65, 95% CI, 1.03–2.65, P=0.039). In addition, adolescent pregnant girls had higher odds of anaemia than their adult pregnant women equivalent (43.9% versus 33.2%; adjusted OR 1.63, 95% CI, 1.01–2.62, P=0.046). Taken together, these data suggest that adolescent pregnant girls were more likely to have malaria and anaemia compared to their adult pregnant counterpart. Results from this study shows that proactive adolescent friendly policies and control programmes for malaria and anaemia are needed in this region in order to protect this vulnerable group of pregnant women.

Prevalence of Malaria Parasitemia among Human Immunodeficiency Virus (HIV) Seropositive Pregnant Women in Kwale, Delta State

A total of 196 pregnant women attending antenatal clinic in Kwale Central Hospital, were prospectively studied to determine their Human immunodeficiency virus status and Malaria parasitaemia from July 2006 to December 2008. Human immunodeficiency virus status and malaria parasitaemia were determined using (WHO) systems two and Field staining technique respectively. Malaria parasite was further confirmed with Geimsa staining technique. Haematocrit and Leishman staining techniques were used for full blood count determination. A prevalence rate of 8.2%,6.6% and 2.6% were observed for HIV, malaria parasite and HIV/ malaria parasite infections among the subjects examined respectively. Also, a prevalence rate of 31.3% was obtained for malaria parasite among HIV seropositive pregnant women with 60% and 40% malaria density respectively. The HIV/malaria positive subjects had reduced mean packed cell volume of 21.2%,mean total white blood cell count of 2900mm/3 and a high absolute neutrophil count. The adoption of routine malaria/HIV screening as parts of management policy to confirm co-infections and proper management of pregnant women in endemic areas is imperative, as discussed on the results of this study.KEYWORDS: Prevalence, Malaria, Human immunodeficiency virus, pregnancy

Role of cyclooxygenase-2 functional gene polymorphisms in Helicobacter pylori induced gastritis and gastric atrophy

In India, the role of host genetic factors is poorly studied for Helicobacter pylori associated diseases. Therefore, we evaluated the association of functionally relevant COX-2 gene polymorphisms (-765 G>C and +8473 T>C) in gastritis and precancerous lesions susceptibility. After upper GI endoscopy, 130 rapid urease test positive patients with non-ulcer dyspepsia, also showed positivity for H. pylori using modified Geimsa staining and anti-CagA IgG serology were included. All patients and 260 asymptomatic controls were genotyped for COX-2 variations using PCR-RFLP. COX-2 -765 (GC+CC) genotypes, -765 C allele, +8473 CC genotype, +8473 (TC+CC) genotypes, +8473 C allele, and variant haplotypes imparted high risk for gastritis (P = 0.036, OR = 1.82; P = 0.007, 1.92; P = 0.025, OR = 2.13; P = 0.017, OR = 1.80; P = 0.017, OR = 1.45; P = 0.010, OR = 2.40; P = 0.023, OR = 1.50 and P = 0.012, OR = 2.20 folds, respectively). In contrast, COX-2 -765 C allele carriers had low risk for lymphocyte (P = 0.020, OR = 0.35), plasma cell infiltrations (P = 0.016, OR = 0.33), and gastric atrophy (GA) development (P = 0.019, OR = 0.35). In conclusion, COX-2 variant allele/genotype/haplotype carriers may be at high risk for gastritis. However, COX-2 -765 C allele carriers may be at low risk for GA development.

A2B Adenosine Receptor Gene Deletion Attenuates Murine Colitis

The A(2B) adenosine receptor (A(2B)AR) is the predominant adenosine receptor expressed in the colonic epithelia. We have previously shown that A(2B)AR mRNA and protein levels are up-regulated during colitis. In this study, we addressed the role of the A(2B)AR in the development of murine colitis and the potential mechanism underlying its effects. Dextran sodium sulfate (DSS), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and Salmonella typhimurium were used to induce colitis in A(2B)AR-null mice (A(2B)AR(-/-)). Colitis was determined using established clinical and histologic scoring. Keratinocyte-derived chemokine (KC) measurements were performed using an enzyme-linked immunosorbent assay. Colonic inflammation induced by DSS, TNBS, or S typhimurium was attenuated in A(2B)AR(-/-) compared with their wild-type counterparts. Clinical features, histologic score, and myeloperoxidase activity were significantly decreased in A(2B)AR(-/-) mice. However, A(2B)AR(-/-) showed increased susceptibility to systemic Salmonella infection. Tissue levels of the neutrophil chemokine, KC was decreased in colitic A(2B)AR(-/-) mice. In addition, flagellin-induced KC levels were attenuated in A(2B)AR(-/-) mice. Neutrophil chemotaxis in response to exogenous interleukin-8 was preserved in A(2B)AR(-/-) mice, suggesting intact neutrophil migration in response to appropriate stimuli. These data demonstrate, for the first time, that the A(2B)AR plays a proinflammatory role in colitis. A(2B) receptor antagonism may be an effective treatment for acute inflammatory intestinal diseases such as acute flare of inflammatory bowel disease.

Correlation Between Serum Lipids Profile With Sperm Parameters of Infertile Men With Abnormal Semen Analysis

Background: In almost 50% of the cases, male factor is involved with infertility. One of the main laboratory tests for evaluation of infertility is semen analysis (SA). However, additional tests may be suggested for further diagnosis of male fertility potentials. The seminal fluid contains sperm, non-sperm cells, and various types of lipids and glucose. Objective: The main objective of this study was to correlate the sperm parameters with concentrations of cholesterol, triglyceride, LDL, and HDL in serum samples of infertile men with abnormal SA. Materials and Methods: A total of 120 infertile men (aged 23–49 years) with abnormal SA were enrolled for this cross-sectional study. Sperm concentration and motility were evaluated using Makler chamber. Normal morphology was done after Geimsa staining. Following 12 h of fasting, the blood samples were obtained for evaluation of cholesterol, triglyceride, LDL, and HDL levels. The lipid profiles were compared with the rates of normal and abnormal sperm parameters. Chi-squared and Fisher exact tests were used for data evaluation. Results: 75.5% and 98% of the subjects with normal levels of triglyceride had abnormal sperm morphology and progressive motility, respectively The levels of LDL and HDL were normal in 80% and 89.3% of the cases, respectively. The majority of the patients with normal LDL had abnormal sperm parameters. Hypertriglyceridemia was observed in 53% of obese men. Also, hypercholesteremia was noticed in 11.8% of overweight and obese individuals. Conclusion: The results showed that the concentrations of serum lipids were not related to the quality of semen parameters. Although obesity did not have any adverse effect on semen parameters, it may be related to sperm quality at cellular or molecular levels. Further studies on the role of lipid profiles of infertile men on sperm fertilizing potentials are recommended.

Characterization of Vaccine Site and Cellular Infiltrate in Subjects with Chronic Myeloid Leukemia (CML) Receiving K562/GM-CSF Vaccination in Combination with Imatinib Mesylate with or without Topical Imiquimod.

Increased understanding of the local injection site infiltrate in response to tumor vaccines may facilitate more effective anti-cancer vaccination strategies. A pilot vaccination strategy was developed to determine if K562/GM-CSF immunotherapy could enhance T cell reactivity and clinical responses in CML patients undergoing therapy with imatinib mesylate. K562/GM-CSF is a tumor vaccine derived from a CML cell line that expresses several defined CML associated antigens and has been genetically modified to secrete GM-CSF. We undertook a correlative project comparing the cellular infiltrates from pre- and post-vaccination skin biopsies in this immunotherapy trial.GM-CSF producing tumor vaccines are effective in recruiting antigen presenting cells (APCs), however alone they are insufficient to initiate APC maturation. Because topical imiquimod (a Toll-like receptor 7 agonist) is known to enhance the in vivo maturation of the recruited APCs, the vaccines (1 x 10*8 cells distributed over 10 injection sites) were given with or without topical 5% imiquimod cream. Imiquimod was applied 4 hrs post-vaccination and then 3d and 5d post-vaccination to injection sites, with at least 1 site left without imiquimod treatment. A series of 4 vaccines were administered in 3 wk intervals. Six mm punch biopsies were taken at baseline, and 3d following the 1st and 4th vaccination. Biopsies were performed at the imiquimod site with the largest area of induration, as well as a site not exposed to imiquimod.Immunohistochemistry of CD3, CD4, CD8, CD1a (Langerhans cell (LC)), factor XIIIa (dermal dendritic cell), and CD68 (monocyte/macrophage) and Geimsa staining was performed. Staining is reported as number of live cells per mm2 in the epidermis and dermis for CD1a+ cells and dermis for the remaining stains. Fifteen subjects agreed to the procedures as part of the clinical trial. Mean area of induration of imiquimod sites was increased significantly compared to the non-imiquimod sites after both the first (p=0.005) and fourth vaccinations (p=0.068). Geimsa staining revealed significant increases in proportion of neutrophils, eosinophils, and mononuclear cells to total number of staining cells after the 1st vaccination in the sites treated with imiquimod compared to the pre-vaccination biopsies while the increases at the sites without imiquimod treatment did not reach statistical significance. We observed increases in CD3+, CD4+, and CD8+ cells at post-vaccination sites. Interestingly, the total number of CD1a+ LCs in the area measured did not appear to be affected by the administration of imiquimod and was constant after vaccinations. However, distribution of CD1a+ LCs shifted from the epidermis to the dermis after vaccination. In addition we observed recruitment of factor XIIIa+ dermal dendritic cells and CD68+ macrophages to the vaccination site that was increased by imiquimod. Epidermal APCs known as LCs migrate to the dermis, yet maintain homeostasis of the total LCs following vaccination independent of Imiquimod application. A correlation across subjects between these histologic features and clinical response to vaccination is on-going.

Genotoxic Effects of Mercury in Channa Punctatus

In order to assess the effect of mercury on the karyotype of Channa punctatus, the fishes were exposed to mercury in the form Mercuric nitrate at various doses (0.019, 0.038 and 0.075 ppm) for 96, 120 and 144 hours. The karyotype was prepared by standard colchicine, potassium chloride, aceto-methanol, air-dried and geimsa staining technique. It was observed that the percentage of Total Metaphase spreads with Chromosomal Aberrations (TMCA) increased with the enhanced mercury levels and exposure time. Mercury induced structural chromosomal aberrations, which included breaks, fragments, dicentric and ring type chromosomes. The study revealed that just 96 hours of exposure to mercury even to.a concentration as low as 0.019 ppm induces chromosomal aberrations in fish.

Basement membrane components enhance isolated enterocyte growth

Isolated enterocyte transplantation may have a potential role in increasing intestinal surface area. However, enterocytes are notoriously difficult to grow. Basement membrane components (BMC) promote adherence, migration, and differentiation of enterocytes. Our aim was to determine if BMC enhance enterocyte growth. Twenty-nine rabbits had 5-cm ileal segments resected and serosal pouches ( n =.22) created from the serosal surface of the colon. Harvesting of enterocytes by warm trypsinization resulted in 92 ± 6% cell viability and yielded 5.0 ± 2.4 10 6 enterocytes/cm intestine. Enterocytes (10 5) were cultured in vitro ( n = 7) in 10 ml growth media in plain flasks and flasks coated with laminin alone or Matrigel (an extract of mouse basement membrane containing laminin plus Type IV collagen and heparan sulfate). The cells were subcultured at 2 weeks and examined after Geimsa staining at 4 weeks. Epithelial growth was confirmed by light microscopy and staining for cytokeratin and quantitated by image analysis (JAVA). Epithelial coverage of the flasks was greater with Matrigel (80 ± 15%) than laminin (66 ± 15%) which was greater than control (44 ± 20%) ( P < 0.01). For in vivo studies 10 5 harvested cells were infused into the serosal pouches either in growth media ( n = 9) or media + Matrigel ( n = 13). Epithelial growth in the pouch was evaluated by qualitative scoring of cytokeratin staining. Cytokeratin staining was similar on control colon serosa ( n = 5) and serosa after infusion of cells in media alone. However, Matrigel significantly enhanced the area, depth, and intensity of staining. These effects were apparent at 1 and 2 weeks, but diminished at 4 weeks. BMC enhance enterocyte growth in vitro. A combination of components had a greater effect than laminin alone. BMC increased cytokeratin staining in serosal pouches suggesting that they also enhance enterocyte growth in vivo.