Smell loss associated with SARS-CoV-2 is not clinically different from other viruses: a multicenter cohort study.

BACKGROUND: Although the COVID-19 pandemic has increased the prevalence of cases with olfactory loss, other respiratory viruses can also cause this condition. We aimed to compare the prevalence of acute SARS-CoV-2 infection and other respiratory viruses in patients with sudden smell loss, and to assess the impact of SARS-CoV-2 viral load and co-infection on olfactory symptoms. METHODS: Patients with sudden smell loss were recruited in a multicenter prospective cohort study in 15 hospitals in Brazil. Clinical questionnaire, Connecticut Chemosensory Clinical Research Center (CCCRC) olfactory test and nasopharyngeal swab to perform a PCR-based respiratory viral panel were collected at first visit (day 0) and 30 and 60 days after recruitment. RESULTS: 188 of 213 patients presented positive test result for SARS-CoV-2, among which 65 were co-infected with other respiratory viruses (e.g., rhinovirus, enterovirus, and parainfluenza). 25 had negative test results for SARS-CoV-2. Patients in both SARSCoV-2 and non-SARS-CoV-2 groups had objective anosmia (less than 2 points according to the psychophysical olfactory CCCRC) at day 0, with no significant difference between them. Both groups had significant smell scores improvement after 30 and 60 days, with no difference between them. Co-infection with other respiratory viruses, and SARS-CoV-2 viral load did not impact olfactory scores. CONCLUSION: Patients with sudden smell loss associated with SARS-CoV-2 and other respiratory viruses had similar presentation, with most participants initiating with anosmia, and total or near total recovery after 60 days. SARS-CoV-2 viral load and co-infections with other respiratory viruses were not associated with poorer olfactory outcomes.

A comparative effectiveness pilot study of teriparatide for medication-related osteonecrosis of the jaw: daily versus weekly administration.

We studied the effectiveness of teriparatide (TPTD) for treating medication-related osteonecrosis of the jaw (MRONJ) in patients with osteoporosis and examined differences in the clinical outcomes following daily versus weekly TPTD. The outcomes were significantly improved in the entire patient series and the daily group. PURPOSE: Teriparatide (TPTD) treatment for Stage II-III medication-related osteonecrosis of the jaw (MRONJ) in osteoporotic patients has yielded promising results in uncontrolled studies. The daily administration and the weekly administration of TPTD have been reported to improve outcomes in MRONJ. Herein, we sought to identify differences in the clinical outcomes of MRONJ patients treated with daily TPTD versus weekly TPTD. METHODS: We enrolled 13 patients and randomly assigned them to receive either of two treatments: 1×/week 56.5-µg TPTD injection for 6 months (weekly group; n = 6 patients after 1 dropout), or 20-µg TPTD injection daily for 6 months (daily group; n = 6 patients). Patients in both groups received conventional therapy plus intensive antibiotic therapy as necessary. We compared the changes in the patients' clinical stage of MRONJ, bone metabolism, percentage of bone formation, and bone turnover markers between the weekly and daily groups. RESULTS: TPTD treatment with MRONJ led to partial remission or complete remission in 5 daily-group patients and 3 weekly-group patients. The MRONJ stage was significantly improved from baseline to 6 months of treatment in the entire series of 12 patients (p = 0.008); the weekly group did not show significant improvement, but the daily group did (p = 0.01). CONCLUSIONS: This study provides the first comparison of clinical outcomes between MRONJ patients who received daily or weekly TPTD injections. Six months of treatment with TPTD realized a significant improvement of MRONJ stage in both the entire patient series and the daily group.

Effect of Lipopolysaccharide on the Progression of Non-Alcoholic Fatty Liver Disease in High Caloric Diet-Fed Mice.

The incidence of non-alcoholic steatohepatitis (NASH) is increasing. Because gut microbiota have been highlighted as one of the key factors in the pathogenesis of metabolic syndrome, we investigated the involvement of the bacterial component in the progression of non-alcoholic fatty liver (NAFL) to NASH. C57BL/6 mice were fed with maintenance food (MF, groups A and B) or a high caloric diet (HCD, groups C and D) for 1 month. Mice were then divided into four groups: Groups A and C were inoculated with PBS, while groups B and D were inoculated with lipopolysaccharide (LPS) plus complete Freund's adjuvant (CFA). The inoculations were performed a total of 3 times over 3 months. At 6 months, while hepatic steatosis was observed in groups C and D, cellular infiltration and fibrosis were less evident in group C than in group D. Inflammatory cytokines were upregulated in groups B and D. 16S rRNA pyrosequencing of whole colon homogenates containing faeces showed that certain bacterial groups, such as Bacteroidaceae, Peptostreptococcaceae and Erysipelotrichaceae, were increased in groups C and D. Although loading of bacterial components (LPS) resulted in hepatic inflammation in both MF- and HCD-fed mice, HCD feeding was more crucial in the progression of NAFL during the triggering phase.

[Lung cancer accompanied by active pulmonary tuberculosis].

We report 2 patients with lung cancer accompanied by active pulmonary tuberculosis. Case1 was a 82-year-old woman with stage I A bronchioloalveolar carcinoma and tuberculosis in right upper lobe. Right upper lobectomy was performed after the histological diagnosis of lung cancer by intraoperative frozen section. Case2 was a 69-year-old man with papillary adenocarcinoma in right lower lobe and tuberculosis in bilateral upper lobe. Partial resection in right lower lobe was performed for diagnosis of lung cancer. Smear-positive tuberculosis was diagnosed by sputum examination after the operation. Post-operative anti-tuberculosis chemotherapy was added in both patients.

Identification of the motility-related protein (MRP-1), recognized by monoclonal antibody M31-15, which inhibits cell motility.

A murine monoclonal antibody (M31-15) was identified using the penetration-inhibiting assay of a human lung adenocarcinoma cell line (MAC10) and remarkably inhibited the phagokinetic tract motility of various cancer cell lines. The antigen, motility-related protein (MRP-1), recognized by M31-15, was 25- and 28-kD proteins, and M31-15 was used to isolate a cDNA clone from a human breast carcinoma cDNA library. Sequence analysis revealed that MRP-1 had strong similarity with a B cell surface antigen (CD37), a melanoma-associated antigen (ME491), the target of an antiproliferative antibody (TAPA-1), a human tumor-associated antigen (CO-029), and the Sm23 antigen of the trematode parasite Schistosoma mansoni.

Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA.

Previously we showed that motility-related protein (MRP-1) is an antigen recognized by monoclonal antibody (mAb) M31-15 inhibiting cell motility and that the sequence of MRP-1 coincides with that of CD9. In the present study, plasmid was constructed in which human MRP-1/CD9 cDNA is expressed under the control of the Abelson murine leukemia virus promoter sequence. The expression plasmid for MRP-1/CD9 was introduced into Chinese hamster ovary cells, human lung adenocarcinoma cell line MAC10 (MRP-1 positive), and human myeloma cell line ARH77 (MRP-1 negative). All of the MRP-1/CD9 (over)expressing clones obtained from these transfected cells showed suppressed cell motility (penetration and phagokinetic track assays) depending on the degree of expression of MRP-1/CD9. Overexpression of MRP-1/CD9 by MAC10 cells resulted in the suppression of cell motility (maximally 73%) associated with considerable inhibition of the cell growth (maximally 48%). However, the inhibition of the growth of MAC10 cells by mAb M31-15 was < 17% at an antibody concentration of 1-5 micrograms/ml, which inhibits cell motility by > 90%. These results suggest that MRP-1/CD9 directly regulates cell motility and may also affect cell growth. Effects on metastasis by the expression of MRP-1 CD9 were investigated with mouse melanoma BL6 cells-BALB/c nu/nu mouse system. Metastatic potential of all transformants expressing MRP-1/CD9 was lower than that of parent BL6 cells.

Morphological study of cationic polymer-anionic surfactant complex precipitated in solution during the dilution process.

We investigated the phase diagrams and the morphology of the complexes that were formed by cationic polymers, cationic cellulose (CC) and cationic dextran (CD), and by anionic surfactant-based sodium poly(oxyethylene) lauryl ether sulfate (LES). The anionic charge of the LES-based surfactants was changed by adding an amphoteric surfactant, lauryl amidopropyl betaine acetate (LPB), or a nonionic surfactant, polyoxyethylene stearyl ether (C18EO25). We discuss the relationship between the complex aggregation process and the morphology of the precipitated complexes. The morphologies of CC complex aggregates, which precipitated during the dilution process in a model shampoo solution, changed from membranous forms to mesh-like forms by decreasing the charges of both the CC and the surfactant. Their touch on hair in the rinsing process changed from sticky to smooth and velvety, corresponding to their rheological properties. In contrast, CD complex aggregates had a membranous form and a smooth touch independently of the charges on the polymer and surfactant. These results suggested that the control of the charges of both the polymer and surfactant and the choice of polymer structure are important for excellent conditioning effects upon rinsing with shampoo.

Application of positron emission tomography in physical medicine.

Positron emission tomography (PET) is widely used in the fields of clinical and basic medicine. The PET device utilizes coincidence logic to detect annihilation photons emitted from positrons and estimates physiological functions of human organs in vivo. Radiopharmaceutical 18F- fluorodeoxyglucose (FDG), an analogue of glucose, is trapped metabolically in cells after being administered into the body, and can be substantially used for evaluating physiological and biochemical functions in vivo. Here, we attempted to describe the basics of PET as well as to apply the technique together with 18F-FDG as a tracer for evaluating organ glucose metabolism induced by exercise. Three-dimensional (3D) FDG-PET was applied to normal volunteers who performed exercise to evaluate whole-body glucose metabolism. Regions of interest analysis were drawn on visually defined regions (i.e., lower limbs, thigh, liver, intestine, brain, heart, etc.) to determine radioactivity distribution. FDG-PET clearly showed the recruitment of energy resources from abdominal organs to lower limb skeletal muscles to balance energy expenditures. The results suggested that 3D FDG-PET can be applied as an imaging tool to physical medicine.

Effects of liquid preservation of sperm on their ability to activate oocytes and initiate preimplantational development after intracytoplasmic sperm injection in the pig.

The objective of this study was to clarify the effects of liquid preservation conditions on the ability of pig sperm to activate oocytes, form a male pronucleus, and initiate preimplantational development of embryos after intracytoplasmic sperm injection (ICSI). Porcine ejaculates were preserved at 4, 14, and 24 degrees C for up to 48h, and then damage to the plasma membrane, morphologic changes of the acrosome, and the amount of phospholipase Czeta (PLCzeta) in the sperm were assessed by SYBR-14/propidium iodide staining, fluorescein isothiocyanate-conjugated peanut agglutinin staining, indirect immunofluorescence, and Western blots, respectively. The proportion of sperm with a disintegrated plasma membrane or damaged acrosome increased in all samples as the duration of preservation increased, although the time courses of the increases varied among preservation temperatures. The immunolocalization and immunoreactivity of PLCzeta in the sperm showed its reduction concurrent with disintegration of the plasma membrane and acrosome. Rates of oocyte activation, male-pronuclear formation, and blastocyst formation after ICSI using sperm preserved for 18h at 24 degrees C (78%, 62%, and 35%, respectively) and for 48h at 14 degrees C (63%, 53%, and 28%, respectively) were significantly higher than those of any other sperm sample. We concluded that the damage to the plasma membrane and acrosome, and a sufficient amount of PLCzeta in the sperm head, enhanced successful oocyte activation, fertilization, and early development of the oocytes after ICSI. Moreover, we inferred that appropriate liquid preservation of sperm improved the efficiency of blastocyst production in vitro after ICSI in pigs.

MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2.

Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (P<0.0001). MRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (P<0.0001). However, no difference was observed in the expression of Arp2, Arp3 or other WASPs. A neutralizing anti-MRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (P<0.0001), and reversed the morphological effects of MRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway.

Recent progress of the characterization of oppositely charged polymer/surfactant complex in dilution deposition system.

A mixture of oppositely charged polymer and surfactants changes the solubilized state, having a complex precipitation region at the composition of electric neutralization. This complex behavior has been applied to surface modification in the fields of health care and cosmetic products such as conditioning shampoos, as a dilution-deposition system in which the polymer/surfactant mixture at the higher surfactant concentration precipitates the insoluble complex by dilution. A large number of studies over many years have revealed the basic coacervation behavior and physicochemical properties of complexes. However, the mechanism by which a precipitated complex performs surface modification is not well understood. The precipitation region and the morphology of precipitated complex that are changed by molecular structure and additives affect the performance. Hydrophilic groups such as the EO unit in polymers and surfactants, the mixing of nonionic or amphoteric surfactant and nonionic polymer, and the addition of low polar solvent influence the complex precipitation region. Furthermore, the morphology of precipitated complex is formed by crosslinking and aggregating among polymers in the dilution process, and characterizes the performance of products. The polymer chain density in precipitated complex is determined by the charges of both the polymer and surfactant micelle and the conformation of polymer. As a result, the morphology of precipitated complexes is changed from a closely packed film to looser meshes, and/or to small particles, and it is possible for the morphology to control the rheological properties and the amount of adsorbed silicone. In the future, further investigation of the relationships between the morphology and performance is needed.

Effect of Gender Differences on Transplant Kidney Function.

BACKGROUND: Transplant kidney function is thought to be affected by sex differences, such as physical conditions including muscle volume, sex hormones, immune responses, and so forth. We examined the effect of sex differences on transplant kidney function. METHODS: The subjects were selected from kidney transplant recipients, who received kidney transplantation on our hospital between January 2000 and August 2015. Cadaveric donors and parent-child pairs with an age difference were excluded, then we included 47 recipients whose sex was different from the sex of the donor. We compared transplant kidney function between male donors and female recipients group (M→F, n = 20) and female donors and male recipients group (F→M, n = 27). RESULTS: Nadir creatinine value was higher in the F→M group than in the M→F group (1.09 mg/dL vs 0.76 mg/dL, P < .0001). The estimated glomerular filtration rate (eGFR) was significantly higher in the M→F group than in the F→M group (66.6 mL/min/1.73 m2 vs 50.1 mL/min/1.73 m2, P = .002), and eGFR ratio (recipient to donor) was significantly higher in the M→F group than in the F→M group (1.13 vs 0.57, P < .0001). Multiple linear regression analysis showed that the only the sex of the recipient was significant prognostic factor of eGFR after renal transplantation (P = .037). CONCLUSIONS: The short-term kidney function of the graft from male to female was better than that of the graft from female to male.

Human IgG3 monoclonal antibody directed to an unbranched repeating type 2 chain (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----R) which is highly expressed in colonic and hepatocellular carcinoma.

Previously established human monoclonal antibodies (MAbs) directed to carbohydrate antigens are essentially all IgM class, and show relatively low affinity and low reactivity at 37 degrees C. We report here the establishment of a human IgG3 MAb displaying high affinity antigen-binding activity at 37 degrees C and efficiently activating cellular cytotoxicity directed to human tumor cell lines expressing the polylactosamine antigen. The IgG3 MAb (MH21-134) reacted with the repeated unbranched polylactosamine structure Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----R, i.e., nLc6, nLc8, etc., but did not react with sialyl 2----3 or 2----6 substituted derivatives at the terminal Gal. This specificity differs from that of several anti-i antibodies, or human anti-i-like MAbs which react with sialyl 2----3 substituted structures. Directly biotinylated MH21-134 antibody was used in immunohistochemical staining of 154 formalin-fixed, paraffin-embedded tissue sections to study distribution of the antigen. High incidence of positive staining was found in colon cancer (11/17; 65%) and hepatocellular carcinoma (8/12; 67%), followed by large cell and squamous cell carcinoma of lung cancer (10/13; 59%, and 14/26; 54%, respectively). TLC immunostaining of glycolipid extracts from a variety of tumor tissues showed the presence of nLc6 and/or nLc8 in over 50% of cases. The antigens nLc6 and nLc8 were found to be absent from normal colonic epithelia, kidney, and pancreas. Only a weak band corresponding to nLc8 and one corresponding to nLc6 were found in liver and spleen, although all these normal tissues, including gastrointestinal epithelia, lung, liver, spleen, erythrocytes, and lymphocytes, were essentially negative on immunohistology. However, the antigen was found to be highly expressed in myelocytes and weakly in bronchial glands of lung and pancreatic duct epithelia. Nevertheless, expression of unsubstituted, unbranched polylactosamine antigen could be an important basis for induction of humoral immune response against certain types of human cancer, despite its limited expression in normal cells.

First establishment of a human monoclonal antibody directed to sulfated glycosphingolipids SM4s-Gal and SM4g, from a patient with lung cancer.

Several human monoclonal antibodies directed to tumor-associated glycolipid antigens have been established, but more than one-half of them react with gangliosides and the others react with neutral glycolipids. We report here the first establishment of a human IgM monoclonal antibody directed to the sulfated glycolipid. This monoclonal antibody, M14-376, did not react with SM3 and SB1a which have a terminal HSO3----3Gal beta 1----R1, but with the simple sulfolipids SM4s-Gal and SM4g which contain a terminal HSO3----3Gal beta 1----O----CH2----R2; however, lyso-SM4s-Gal and lyso-SM4g did not bind M14-376. These results suggest that terminal HSO3----3Gal and part of the hydrophobic region of the glycolipid are recognized by M14-376. Directly biotinylated M14-376 was used for immunohistochemical staining of 140 formalin-fixed, paraffin-embedded lung cancer tissue sections to study the distribution of the antigen. A high incidence of positive staining was found in adenocarcinoma (39.5%, 17 of 43), followed by large cell carcinoma (20.0%, 5 of 25), while this antigen was rarely detected in small cell carcinoma (4.7%, 1 of 21) and squamous cell carcinoma (3.9%, 2 of 51). Thin layer chromatography immunostaining of glycolipids extracted from lung cancer tissues showed the presence of only SM4s-Gal in adenocarcinoma, but SM4g was not found in any subtype of lung cancer. Immunohistochemical staining revealed that this antigen was expressed in normal kidney, testis, and brain, but erythrocytes, granulocytes, and lymphocytes were negative in cytofluorometric analysis.

Stage-specific expression of SSEA-1-related antigens in the developing lung of human embryos and its relation to the distribution of these antigens in lung cancers.

The localization of three carbohydrate antigens, Lex, Ley, and sialylated Lex-i, which are closely related to stage-specific embryonic antigen 1, in the lung of developing human embryos was investigated using specific monoclonal antibodies. In the 38-day-old embryo, when the primitive lung bud has appeared and developed into two lung sacs, only Ley antigen was specifically positive in the proliferating cells in the terminal portion of lung bud. In the 50-53-day-old embryos, the future bronchi were actively developing from the bronchial buds. At this stage, the Ley antigen was maximally expressed and the Lex antigen appeared in the bud cells. In the lung of the 12-week-old embryo, buds for the future bronchioles were lined by simple cuboidal epithelial cells, which were strongly positive for Lex antigen, weakly positive for Ley antigen, and still completely negative for sialylated Lex-i antigen. Sialylated Lex-i antigen finally appeared in 18-week-old embryos, in the cells of the terminal buds for the future alveoli. At this stage, the Lex and Ley antigens were already beginning to disappear and were only weakly positive in cells of terminal buds. At 20 weeks, only sialylated Lex-i antigen was weakly detected in the cells in the terminal buds; after 8 months, all three antigens were essentially not detected in the respiratory cells in most of the embryos examined in this study. Formation of bronchial glands was detected at 18 weeks, where the developing gland cells were specifically positive for sialylated Lex-i antigen. Ciliation of the bronchial epithelial cells started at 12 weeks and propagated thereafter. The ciliation was accompanied by the reappearance of Ley and Lex antigen in the epithelial cells. These findings collectively indicated that the three antigens all have a physiological significance as stage-specific developmental antigens of the human lung; those antigens were specifically present in the bud cells at each important step of the morphogenesis of the human lung, such as cells in the lung buds, bronchial buds, and terminal buds for the formation of the alveolus, and cells differentiating into bronchial gland cells. The three antigens gradually disappear in the later stage of development along with the maturation process of the lung. Stage-specific embryonic antigen 1 and related antigens are known to be associated with various human cancers, including lung cancers. We suggest that the expression of these antigens in the lung cancer cells is the result of the retrodifferentiation of the cancer cells to the stages of immature embryonic lung cells.

Motility-related protein-1 (MRP-1/CD9) reduction as a factor of poor prognosis in breast cancer.

The application of reliable markers is of major importance for predicting the prognosis of and instituting the appropriate postsurgical treatment of patients with breast cancer. Previously we showed that motility-related protein-1 (MRP-1), which is identical to CD9, regulates cell motility, and that cultured tumor cells transfected with MRP-1/CD9 cDNA have low motility and low metastatic potential. In addition, MRP-1/CD9 immunoblotting and immunohistochemical study with breast cancer revealed that MRP-1/CD9 expression diminished as the clinical stage of a given breast cancer advanced and that the MRP-1/CD9 gene and protein expression in the metastatic lymph nodes was strikingly lower than in the primary breast cancers. In this study, we also investigated the expression of MRP-1/CD9 by immunoblotting and immunohistochemical analysis in 143 freshly resected invasive ductal carcinomas of the breast: 52 tumors were stage I, 61 were stage II, and 30 were stage III. Tumors were classified as MRP-1/CD9 positive when a band intensity of >30% compared with positive control cells, ZR-75-30 were evaluated with the antibody M31-15, and those with intensities <30% as negative. Moreover, these results were ascertained by immunostaining. Tumor specimens classified as MRP-1/CD9 positive using Western blotting had >50% of the cancer cells immunostained with M31-15, and those classified as MRP-1/CD9 reduced had <50% of the cancer cells immunostained with M31-15. There were 97 patients with MRP-1/CD9 positive tumors and 46 patients whose tumors had reduced MRP-1/CD9 levels. The disease-free rate of the former group of patients was strikingly higher than that of the latter (84.7% versus 51.4%, P<0.001). Similarly, the overall survival rate was also significantly different between the two groups (93.6% versus 69.6%, P=0.004). Multivariate analysis with the Cox regression model indicated that MRP-1/CD9 positively correlated better with disease-free survival (P<0.001) than estrogen receptor, tumor, and lymph node status. Our data suggest that low MRP-1/CD9 expression by tumors of the breast may be associated with poor prognosis. It is conceivable that testing for MRP-1/CD9 may identify node-negative breast cancer patients who are at high risk for early disease recurrence.

Motility related protein 1 (MRP-1/CD9) expression: inverse correlation with metastases in breast cancer.

In our previous studies we showed that motility related protein 1 (MRP-1) is a glycoprotein recognized by mAb M31-15, and that the sequence of MRP-1 is identical to that of CD9, a WBC differentiation antigen. Transfection of MRP-1/CD9 cDNA into cultured nonhematopoietic cells suppresses cell motility. The extent of suppression is directly related to the level of MRP-1/CD9 expression. In addition, the metastatic potential of MRP-1/CD9-transfected melanoma BL6 cells is lower than that of control BL6 cells. To determine whether these experimental results are of relevance with respect to actual human tumors, we investigated MRP-1/CD9 expression in 143 invasive ductal carcinomas of the breast. Of 97 patients with MRP-1/CD9-positive tumors, only 36 (37.1%) had lymph node involvement. In contrast, 21 of 39 (53.8%) patients whose tumors had reduced MRP-1/CD9 immunoreactivity and 5 of 7 patients whose primary carcinomas were not stained by the anti-MRP-1/CD9 MAb had lymph node metastases. The comparison of protein expression by 62 primary tumors and their respective metastatic lymph nodes revealed that in almost 50% of the cases, the latter had lower MRP-1/CD9 levels than the former. Moreover, reverse transcriptase-PCR-based analysis disclosed that MRP-1/CD9 gene expression in the metastatic lymph nodes of 17 of 32 patients was strikingly lower than in the primary invasive ductal carcinomas. Gene overexpression was not observed in any of the samples studied. Our data suggest that low MRP-1/CD9 expression may be associated with the metastatic potential of certain human tumors.

Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small cell lung cancer.

As part of our evaluation of members of the transmembrane 4 super-family as possible prognostic predictors, we performed a retrospective study on the expression of the recently identified KAI1 gene by tumors of the lung. This gene, which is identical to CD82, suppresses tumor metastasis of prostate cancer, and its decreased expression may be involved in malignant progression. We used reverse transcription-PCR to analyze tumor tissues from 151 lung cancer patients; 74 tumors were stage I, 17 were stage II, and 60 were stage III. Our results indicate that while 35 patients had tumors in which the KAI1/CD82 gene was conserved (positive), 116 patients had tumors with reduced gene expression (negative). The overall survival rate of patients with KAI1/CD82-positive tumors was significantly higher than that of patients with KAI1/CD82-negative tumors (77.4% versus 38.5%; P=0.002). Furthermore, the overall survival rate of patients with KAI1/CD82-positive adenocarcinoma was also much higher than that of individuals whose adenocarcinoma had reduced KAI1/CD82 expression (73.4% versus 27.1%;P=0.009). Multivariate analysis with the Cox regression model indicated that KAII/CD82 positivity correlated best with the overall survival rate, except for lymph node status. Our data suggest that high KAII/CD82 gene expression by tumors of the lung may be associated with a good prognosis. These findings complement our earlier studies on MRP-1/CD9, another member of the transmembrane 4 superfamily, whose reduced expression in non-small cell lung cancer appears to be a factor of poor prognosis. This set of observations suggests that assessment of the expression status of KAI1/CD82 and MRP-1/CD9 by tumors may provide prognostic information on the clinical behavior of lung cancer.

Generation of two murine monoclonal antibodies that can discriminate N-glycolyl and N-acetyl neuraminic acid residues of GM2 gangliosides.

Since the preliminary analyses of the glycolipids of small cell carcinomas of the lung showed an increase of GM2 ganglioside, we generated new murine monoclonal antibodies directed to GM2 to identify the molecular species of the glycolipid. The monoclonal antibodies MK2-34 and MK1-16 (both IgM), which specifically detect N-glycolyl GM2 and N-acetyl GM2, respectively, were generated by immunizing mice with liposomes containing monophosphoryl lipid A, trehalose dimycolate, and the antigenic ganglioside. Among the glycolipid preparations extracted from the cancer tissues of 39 patients with lung cancer, a significant amount of N-acetyl GM2 was detected with MK1-16 antibody in 70% of the squamous cell carcinoma cases, 50% of the lung adenocarcinoma cases, 33% of the large cell carcinoma cases, and 100% of the cases of small cell carcinoma of the lung. On the other hand, N-glycolyl GM2 which was defined by the monoclonal MK2-34 was not found in any of the glycolipid fractions prepared from the lung cancer tissues examined in this study. Immunohistochemical studies of the lung cancer tissues with the MK1-16 antibody showed that the N-acetyl GM2 was present not only in small cell carcinoma tissues as one of the antigens related to tumors of neuroectodermal origin, but also in the squamous cell carcinoma and adenocarcinoma of the lung with a comparable frequency. The appearance of the N-acetyl GM2 antigen correlated well with the degree of differentiation of the cancer cells in patients with squamous cell carcinoma and adenocarcinoma of the lung.