The Prognostic Value of the Preoperative Lymphocyte Count in Patients with Gastric Cancer

PURPOSE: The aim of this study was to evaluate the prognostic value of the peripheral blood lymphocyte count before surgery in those patients with gastric cancer. MATERIALS AND METHODS: The study group was comprised of a series of 1,054 patients who underwent curative gastrectomy. The appropriate lymphocyte count cutoff value was determined. The prognostic factors were evaluated by univariate and multivariate analyses. RESULTS: The lymphocyte count cutoff value was 1,500/ul. The patients were classified into two groups: Group A had a lymphocyte count > or =1,500/ul (n=765) and Group B had a lymphocyte count <1,500/ul (n=289). There were statistically significant differences between the groups according to their age (P<0.001), the tumor stage (P=0.038) and the tumor size (P<0.001). The 5- and 10-year survival rates of Group A were 80.1% and 76.6%, respectively and those of Group B were 72.4% and 63.5%, respectively (P=0.002). When multivariate analysis was performed by the Cox proportional hazards model, the lymphocyte count was not an independent prognostic factor. CONCLUSION: Although the prognosis of patients with a high lymphocyte count was better than that of the patients with a low lymphocyte count, our results did not support using the preoperative peripheral blood lymphocyte count as an independent prognostic factor for patients with gastric cancer.

Clinical Features and Prognostic Factors Influencing Long-term Survival in pT2 Gallbladder Carcinoma Patients

PURPOSE: The prognosis of gallbladder carcinoma is unfavorable, and the depth of invasion, the T-factor, is the most important prognostic factor. T2 tumors are not easily diagnosed preoperatively, and they have unpredictable behavior. In this study, we reviewed the clinicopathologic features of pT2 gallbladder carcinoma and identified prognostic factors. METHODS: We enrolled 64 pT2 gallbladder carcinoma patients who underwent surgery in Kyungpook National University Hospital between January 1992 and July 2006. The clinicopathologic features were retrospectively reviewed, and univariate and multivariate analyses were performed using the Kaplan-Meyer method and Cox regression hazard model to identify factors influencing long-term survival. RESULTS: Twenty-one patients (32.8%) were asymptomatic, with seven tumors (10.9%) found incidentally. The gallbladder body was the most commonly involved site (45.3%, 29/64). Although CA19-9 was not sensitive, it was helpful for predicting recurrence. The 5-year diseasespecific survival rate in R0 resection was 75.9%, whereas no patients in the R1 or R2 resection groups survived more than 18 months.ANone of the clinicopathologic factors was prognostic. The unfavorable prognostic effect associated with lymph node involvement highlights the importance of complete lymph node dissection. Partial liver resection favorably affected survival, but not to a statistically significant degree. CONCLUSION: Absence of symptoms and specific imaging findings in patients with considerable pT2 gallbladder carcinoma warrants high disease suspicion, and every effort should be taken to achieve R0 resection with extensive lymph node dissection, since it may improve long-term survival in pT2 carcinoma patients.

The Characteristics of Eosinophilc Lung Diseases Cause by Toxocara Canis Larval Infestation / 결핵및호흡기질환

BACKGROUND: Toxocariasis is a common cause of eosinophilia and eosinophilic lung disease in Korea. We analyzed the characteristics of eosinophilic lung disease in toxocariasis. METHOD: One hundred and forty one patients with eosinophilia caused by a toxocara larval infection were evaluated from September 1, 2001 through March 30, 2006. The plain chest x-ray, chest CT(computed tomography), and bronchoalveolar larvage(BAL) were examined. A diagnosis of toxocariasis was made by ELISA using that secretory-excretory antigen from the T. canis larvae. RESULTS: Toxocarial eosinophilic lung diseases was diagnosed in 32 out of 141 patients. Ground glass attenuation was the main feature on the CT scans in 23 out of 141 patients (71.9%). Thirteen patients (40.6%) had a random in zonal distribution on CT. Pleural effusion was observed in 9 patients (28.1%). Twenty eight patients (87.5%) complained of respiratory symptoms. Eleven patients (34.4%) had gastrointestinal symptoms and 12 patients (37.5%) had liver infiltration. CONCLUSIONS: The most common findings of the chest CT in patients with toxocariasis was a randomly distributed ground grass attenuation. A toxocara infection should be considered in a differential diagnosis of patients who exhibit pulmonary infiltration with eosinophilia in Korea.

A human infection of Echinostoma hortense in duodenal bulb diagnosed by endoscopy

As gastroduodenoscopy performed more frequently, case reports of human echinostomiasis are increasing in Korea. A Korean woman presented at a local clinic with complaints of abdominal pain and discomfort that had persisted for 2 weeks. Under gastroduodenoscopy, two motile flukes were found attached on the duodenal bulb, and retrieved with endoscopic forceps. She had history of eating raw frog meat. The two flukes were identified as Echinostoma hortense by egg morphology, 27 collar spines with 4 end-group spines, and surface ultrastructural characters. This report may prove frogs to be a source of human echinostome infections.

Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.

Ultrastructural localization of 28 kDa glutathione S-transferase in adult Clonorchis sinensis

Glutathione S-transferase (28GST) with molecular mass of 28 kDa is an antioxidant enzyme abundant in Clonorchis sinensis. In adult C. sinensis, 28GST was localized in tegumental syncytium, cytons, parenchyma, and sperm tails examined by immunoelectron microscopy. C. sinensis 28GST was earlier found to neutralize bioreactive compounds and to be rich in eggs. Accordingly, it is suggested that 28GST plays important roles in phase II defense system and physiological roles in worm fecundity of C. sinensis.

Molecular cloning and characterization of an antigenic protein with a repeating region from Clonorchis sinensis

In the course of immunoscreening of Clonorchis sinensis cDNA library, a cDNA CsRP12 containing a tandem repeat was isolated. The cDNA CsRP12 encodes two putative peptides of open reading frames (ORFs) 1 and 2 (CsRP12-1 and -2). The repetitive region is composed of 15 repeats of 10 amino acids. Of the two putative peptides, CsRP12-1 was proline-rich and found to have homologues in several organisms. Recombinant proteins of the putative peptides were bacterially produced and purified by an affinity chromatography. Recombinant CsRP12-1 protein was recognized by sera of clonorchiasis patients and experimental rabbits, but recombinant CsRP12-2 was not. One of the putative peptide, CsRP12-1, is designated CsPRA, proline-rich antigen of C. sinensis. Both the C-termini of CsRP12-1 and -2 were bacterially produced and analysed to show no antigenicity. Recombinant CsPRA protein showed high sensitivity and specificity. In experimental rabbits, IgG antibodies to CsPRA was produced between 4 and 8 weeks after the infection and decreased thereafter over one year. These results indicate that CsPRA is equivalent to a natural protein and a useful antigenic protein for serodiagnosis of human clonorchiasis.

Antigenic protein fractions reacting with sera of sparganosis patients

To observe the antigenic protein fractions in saline extract of Spirometra mansoni plerocercoid (sparganum), the crude extract was separated in reducing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The proteins, transferred by electrophoresis to nitrocellulose paper, were reacted with sera from 15 surgically confirmed sparganosis and 24 cysticercosis patients for immunoblotting. Out of 30 identified protein bands in the extract, bands of 29 and 36 kilodaltons (kDa) were the strongest and the most frequently reacting with specific antibody (IgG) in sparganosis sera. Bands of higher molecular weight also reacted with the sera but their frequency of reactions was lower. Sera of cysticercosis reacted with different protein bands in saline extract of sparganum, but the cross reactions were observed in strong antigenic bands of 29 and 36 kDa.

Biochemical properties of a purified protein in cystic fluid of Taenia solium metacestodes

By affinity chromatography using a monoclonal antibody as ligand, Kim et al. (1986) purified a protein fraction in cystic fluid of Taenia solium metacestodes (CF). In this study, the biochemical properties of the purified protein were characterized. Discontinuous-polyacrylamide gel electrophoresis (disc-PAGE) of the protein at 4.5~10% separating gel concentration showed its molecular weight (MW) to be 150 kilodalton(kDa) in non-denatured state, while denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that it was composed of 3 different subunits with respective MW of 15, 10 and 7 kDa. Subunit of 7 kDa was shown to be linked to other subunits by disulfide bonds. Isoelectric point of the protein was pH 6.8. The protein was relatively heat-stable for immunologic analysis. These properties indicated that the protein, comprising about 70% of total content in CF, had similar biochemical characters with antigen B of Oriol et al.(1971) in hydatid cyst fluid (HF)

Immunoblot observation of antigenic protein fractions in Paragonimus westermani reacting with human patients sera

: In order to observe the antigenic fractions in saline extract of adult Paragonimus westermani, proteins in the crude extract were separated by sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) in reducing conditions. The separated protein fractions were transferred to nitrocellulose paper on which 20 sera from human paragonimiasis were reacted and immunoblotted. Out of 15 stained protein bands in SDS-PAGE, 7 reacted with the sera. Of 14 reacted bands, 30 kilodalton(kDa) band was the most frequently reacted (95%) and was a strong antigen. Protein bands of 23 and 46 kDa were also strong antigens. Bands of over 150 kDa, 120 kDa, 92 kDa, 86 kDa, 74 kDa, 62 kDa, 51 kDa, 32 kDa, 28 kDa, 16.5 kDa and 15.5 kDa were also reactive but their frequencies of the reaction were variable.

Intracranial synthesis of specific IgG antibody in cerebrospinal fluid of neurocysticercosis patients

To determine the source of Cysticercus-specific IgG antibody in cerebro-spinal fluid(CSF), paired samples of serum and CSF were collected from confirmed neurocysticercosis, other neurologic diseases and normal control. The antibody levels in serum and CSF were measured by enzyme-linked immunosorbent assay (ELISA). With the measurement of total protein, albumin and IgG concentration in serum and CSF, the contribution of IgG in CSF were calculated in transudation, exudation and intracranial synthesis using the formula of Tourtellotte and Ma (1978). Mean concentrations of total protein, albumin, IgG and proportional IgG levels in CSF by transudation, exudation and intracranial synthesis were elevated in neurocysticercosis. But only the intracranial synthesis of IgG showed a statistically significant correlation with the specific IgG antibody levels in CSF. In CSF from lateral ventricle in the 4th ventricular neurocysticercosis, the protein concentrations were normal and the specific antibody levels were negative. However, in consecutively secured lumbar CSF from the same patients, the former were increased and the latter were positive. These results indicated that, in neurocysticercosis, the specific IgG antibody in CSF was a local product of intracranial synthesis.

Specific IgG antibody responses in experimental cat metagonimiasis

In order to observe the feasibility of serologic diagnosis of metagonimiasis, saline extracts of metacercariae and 4-week old adults were prepared. Sera from 25 experimentally infected cats were collected from 3 days to 12 weeks after infection. Their levels of specific IgG antibody were measured by ELISA together with 3 sera from non-infected cats. Specific IgG antibody levels began to rise in 7 days after infection, reached their peak in 2-4 weeks and made a plateau thereafter. Cats infected with hundreds of adult worms showed minimal rise of the antibody level. Adult antigen was superior to metacercarial antigen in detecting the specific IgG antibody.

Analysis of antigen specificity using monoclonal and polyclonal antibodies to Cysticercus cellulosae by enzyme-linked immunoelectrotransfer blot technique

To analyse the antigen specificity of patients sera from 24 confirmed neurocysticercosis and a monoclonal antibody, SDS-PAGE using 10-15% linear gradient gel and EITB were done. Cystic fluid, saline extracts of scolex and of whole worm of C. cellulosae, saline extracts of sparganum, hydatid cyst fluid, saline extracts of Fasciola, Clonorchis and Paragonimus were used as antigen. Of protein bands in cystic fluid of C. cellulosae, patient sera reacted frequently to bands of 152, 94, 64, 48, 24, 15, 10 and 7 kDa proteins. To saline extracts of scolex and whole worm of C. cellulosae, patients sera reacted frequently to 94, 64, 52, 39, 34, 15 and 10 kDa bands. Two bands in sparganum extract (130 and 64 kDa) and two bands in hydatid cyst fluid (52 and 27 kDa) were cross-reacting bands with sera from cysticercosis patient. Saline extracts of Fasciola, Clonorchis and Paragonimus did not exhibit cross-reacting bands. Monoclonal antibody to cystic fluid of C. cellulosae was found to react with low molecular weight proteins of 15, 10 and 7 kDa.

Evaluation of enzyme-linked immunosorbent assay in serological diagnosis of human neurocysticercosis using paired samples of serum and cerebrospinal fluid

The applicability of micro-ELISA was evaluatd in human neuro-cysticercosis using paired samples of serum and CSF. A total of 355 cases who were mostly neurologic patients was subjected. Cystic fluid of C. cellulosae was used as antigen in protein concentration of 2.5 micro-g/ml. Serum was diluted to 1:100 and CSF was undiluted in the assay for the specific IgG antibody level. The differential criterion of the positive reaction was the abs. of 0.18 in both samples. The results were summarized as follows: The overall sensitivity of the micro-ELISA in 71 confirmed neurocysticercosis was 90.1%; the sensitivity by serum was 77.5% and that by CSF was 83.1%. CSF was a more sensitive and valuable material. Most of the false negative cases of neuro-cysticercosis showed far lower level of abs. rather than marginal. The overall specificity of the micro-ELISA in 52 confirmed other neurologic diseases was 88.5% ; the specificities by serum and by CSF were 94.2% respectively. Cases of other neurologic diseases did not show false positive reactions in both samples. When serum was assayed, taeniasis(2/18), sparganosis(2/20), paragonimiasis(1/56), clonorchiasis(1/15) and fascioliasis(1/1) cases showed cross reactions. When CSF was assayed, 2 of 10 neuro-sparganosis showed cross reactions while none of 9 neuro-paragonimiasis showed it. Out of 71 confirmed neuro-cysticercosis cases, 6 and 11 showed cross reactions by serum and CSF to crude extract antigen of sparganum; but no case did show it to crude extract antigen of Paragonimus westermani. Ventricular CSF showed low or negative levels of IgG antibody than lumbar CSF unless the lesion was at the lateral ventricle itself. Out of 4 racemose cysticercosis cases, 3 showed positive reaction in serum while all of 3 examined CSF were positive. The above results indicated that the serological test for detecting the specific IgG antibody by micro-ELISA using paired samples of serum and CSF was very helpful for clinical differentiation of neuro-cysticercosis from neurologic diseases of other causes.

Antigenicity of the soluble egg antigen of Paragonimus westermani

To evaluate the immature eggs of Paragonimus westermani as a source of diagnostic antigen, about a million eggs which were excreted by 104 adult worms were collected; their saline extract (soluble egg antigen; PwSEA) was prepared. The specific IgG and IgM antibody levels were observed in experimental dog paragonimiasis by micro-ELISA, using PwSEA as well as whole worm extract of 12 week-old P. westermani (PwWWE). The protein composition of the PwSEA was observed by disc-PAGE. The results could be summarized as follows: Specific IgG antibody to PwSEA began to increase on 8 weeks after the experimental infection; it maintained its high level until the observation period of 13 weeks. The levels of IgM antibody to PwSEA, however, did not show any significant change. Specific IgG antibody to PwWWE began to increase earlier from 2 weeks after the infection and continued to increase until the observation period of 13 weeks. Its level was much higher than that to PwSEA. Specific IgM antibody to PwWWE increased temporarily during 2-8 weeks after the infection. By disc-PAGE, PwSEA showed 2 protein bands of very low motility. The bands of PwSEA corresponded to the first and second bands in the electrophoretic pattern of PwWWE of the 12 week-old worms. The above results indicated that the PwSEA induced antibody production in dog paragonimiasis but its antigenicity was weaker than PwWWE to be used as a diagnostic antigen.

Purification of cystic fluid antigen of Taenia solium metacestodes by affinity chromatography using monoclonal antibody and its antigenic characterization

This study was undertaken to purify cystic fluid (CF) antigen of Taenia solium metacestodes by affinity chromatography using specific monoclonal antibody (McAb) and to characterize the antigenicity of the purified antigen. The hybridoma cell lines, prepared by fusion between mouse plasmacytoma and spleen cells from BALB/c mice immunized with CF, secrected antibodies reacting to various helminthic antigens. Majority of cell lines reacted to CF only but some also reacted to parenchymal antigen of T. solium metacestodes, adult T. saginata, sparganum, hydatid cystic fluid, Paragonimus westermani and Clonorchis sinensis, either in combination with CF, other antigens or independently. Cloned cells derived from monoclonal lines also produced antibodies reacting either to CF only or to other helminthes in combination or independently. These results indicated that CF of T. solium metacestodes contained proteins which possessed antigenic determinants not only specific to CF but also cross reactive with the afore-mentioned helminthes. CF of T. solium metacestodes was purified by affinity chromatography using the McAb which reacted to CF and parenchymal antigens. The affinity-purified antigen(A-Ag) and unbound pool(U-Ag) were separated. A-Ag showed 2 protein bands by disc-PAGE whereas CF exhibited 6 bands and U-Ag consisted of all bands CF had. The diagnostic significance of A-Ag was evaluated by ELISA in human neurocysticercosis and other helminthic and neurologic diseases. By A-Ag, the levels of the specific IgG antibody, as shown by absorbance in sera and CSF, were lower than those of CF and U-Ag. Accordingly, the sensitiviy was about 70 % of CF and U-Ag. However, the nonspecific positive reactions to CF and U-Ag, observed in sparganosis, T. saginata infection and paragonimiasis did not occur when A-Ag was used. These results indicated that the affinity-purified A-Ag had the higher specificity but the lower sensitivity as a diagnostic antigen in cysticercosis, probably because it only detected a single or limited numbers of monospecific antibodies among the diverse polyclonal antibodies produced in the patients with neurocysticercosis.

Serologic follow-up study in neurocysticercosis patients by ELISA after praziquantel treatment

/A total of 69 patients of confirmed neurocysticercosis was followed serologically by ELISA up to 22 months after praziquantel treatment. The intervals and numbers of follow-up were variable by patients. Serially collected samples of serum and CSF were examined simultaneously for their specific IgG antibody levels by ELISA, using cystic fluid, saline extracts of bladder wall and scolex as antigen. Within 4 months after praziquantel treatment, the antibody levels were elevated temporarily in both serum and CSF in most patients. In some cases antibody levels exhibited steady declining tendency after the treatment. Concomitant administration of dexamethasone appeared to suppress the elevation of antibody levels. The rate of mean absorbance of antibody changed more in serum than in CSF. The rate of elevation was greater in antibodies to parenchymal antigens than that to cystic fluid, but absolute difference of antibody levels was greater in anitbody to cystic fluid. Previously negative samples for IgG antibody may become positive after praziquantel treatment, which could be used as a complementary tool(provocation test) in serodiagnosis. One month was considered to be sufficient interval for the follow-up test for that purpose. In the follow-up of up to 22 months, only few cases of chronic neurocysticercosis showed declining tendency of IgG antibody levels below negative range. During acute encephalitic attacks in chronic patients, IgG antibody to parenchymal antigen were elevated in CSF temporarily. These results indicated that serologic follow-up of every year was recommendable to differentiate the cured patients from chronic patients with slowly calcifying lesions.

Electrophoretic patterns of proteins from Paragonimus westermani in early developmental stages

In order to observe the protein compositions of soluble extracts of P. westermani, and their changes during early developmental stages, the crude saline extracts of 4, 6, 8, 10 and 12 week-old worms which were harvested from experimentally infected dogs were analysed by disc-PAGE. The results were as follows: A total of 15 bands were identified from electrophoregrams of respective developmental stages. Of them, 5 bands were recognized throughout the developmental stages. The number and protein amount of identified bands changed according to the worm development from 4 weeks to 12 weeks. However, the banding patterns of 4 and 6 week-old worms and 8 and 10 week-old worms were similar each other. Of 15 identified bands, band 1 was recognized only in 12 week-old worms whereas bands 3, 4, 8, 9, 10, 11 and 15 gradually lowered their amount according to development to disappear in 12 week-old. In addition, band 5 became a major band in 12 week-old while band 6 turned to a minor band at tha same age. The possible relations of changing patterns of protein bands with worm development were discussed.

In situ posture of anterior body of Metagonimus yokogawai in experimentally infected dog

The in situ posture of anterior body of Metagonimus yokogawai was observed in experimental metagonimiasis of dog. The metacercariae were collected from naturally infected sweetfish by peptic digestion; a total of 8 dogs was orally infected with 10,000 metacercariae respectively. Two dogs were killed on 3 days, 9 days, 4 weeks and 10 weeks after the infection. The postures of worms in histological section of small intestine and of whole worms collected from the fixed intestinal mucosa were examined by light and scanning electron microscope. The results were summarized as follows: The recovery rates of worms were 42.6% on 3 days, 55.0% on 9 days, 33.2% on 4 weeks and 9.8% on 10 weeks after the infection respecively. In histological sections of small intesine, most of worms were found at intervillous spaces as ovoid sections of posterior body. However, many worms, especially in 3 day-old worms, revealed protruded anterior body in glandular lumens of crypt. Some sections of anterior body were bifurcated or sacculated. The worms collected from fixed intestinal mucosa under dissecting microscope exhibited a variety of postures. Many worms showed flat shapes with a concavity or curvatures. However, in many worms, the anterior body made a single or multiple protrusions. By SEM observation of protruded anterior body, a longitudinal groove was found. The frequency of worms with protuded anterior body decreased in 9 days, 4 weeks and 10 weeks than in 3 day-old worms. The above findings indicated that the anterior body of juvenile and adult M. yokogawai protruded to lumens of glandular crypt by folding their lateral protions to make a reversible tubelike structure. Frequent multiple protrusions were considered to be made to adapt the microniche of glandular crypts of dog intestine.

Seasonal variations of metacercarial density of Clonorchis sinensis in fish intermediate host, Pseudorasbora parva

The seasonal variations of the rate and intensity of metacercarial infection of C. sinensis in P. parva were observed. The fish were collected at Sun-Am river which located in Kim-Hae City, Kyong-Sang-Nam Do(=Province), Korea, from March 1983 to February 1984 every month. A total of 788 fish was examined. The number of metacercariae in each fish was individually counted after the individual digestion by artificial gastric juice. The result was as follows: During one year, 513(65.1%) out of 788 fish were infected with metacercariae. In May, June, July and September, the infection rates ranged from 82. 0 % to 98. 6% whereas the rates was relatively low in March, April, November and February raning from 11. 4% to 64.7%. The intensity of infection was similar with those of infection rates. The mean intensity per infected fish was 103.0 and standard deviation was 118.9 throughout one year. The highest mean intenstiy was in June(294. 8) and the lowest in Novebmver(11.1). The observed frequency of fish with certain intensities of metcercariae were fitted to theoretical equations derived from negative binomial distribution in March, April, November and February(p>0.05). Meanwhile, the equation of lognormal distribution were fitted with the observed frequencies in May, June, July and September(p>0.05, p>0.75). The variance/mean ratio varied by month. The value was the highest in July(814.3) and the lowest in November(158.8). Unlike our hypothesis, the metacercarial density of Clonorchis sinensis in its the most favourable fish host, Pseudorasbora parva showed considerable seasonal variations in the hyperendemic area. The possible factors were discussed.