Effect of posterior tibial slope on the short-term outcome in mobile-bearing unicompartmental knee arthroplasty / 北京大学学报(医学版)

OBJECTIVE@#To evaluate the relationship between postoperative knee function and the sagittal position of tibial component in unicompartmental knee arthroplasty (UKA).@*METHODS@#We retrospectively enrolled the patients who underwent UKA from January 2016 to May 2020. They were assigned into 2 groups according to postoperative posterior tibial slope (PTS): the normal PTS group (PTS≥3° and PTS < 8°) and the abnormal PTS group (PTS < 3° or ≥8°). The patients were followed up for at least 12 months. The postoperative Knee Society Clinical Score (KSS-C), Knee Society Functional Score (KSS-F) and knee range of motion (ROM) were compared between the two groups.@*RESULTS@#A total of 72 patients (82 knees) were included with 51 patients (58 knees) in PTS normal group and 21 patients (24 knees) in PTS abnormal group. All the patients were followed up with median of 23.6 months. There was no significant difference in the general data [gender, age, body mass index (BMI)], pre-operative knee range of motion, preoperative KSS-C score and KSS-F score (P > 0.01). The KSS-C score, KSS-F score, and knee range of motion significantly improved after surgery (P < 0.01) for all the patients. The postoperative KSS-C score in normal PTS group (88.76±2.79) was significantly higher than the KSS-C score in abnormal PTS group (84.42±3.35, P < 0.01), but no significant difference between the 2 groups was observed in postoperative KSS-F score and knee range of motion (P > 0.01). In addition, there was no correlation between the change of PTS and postoperative KSS-C score (r=-0.034, 95%CI: -0.247 to 0.186, P = 0.759), KSS-F score (r = -0.014, 95%CI: -0.238 to 0.198, P = 0.901) and knee range of motion (r= 0.045, 95%CI: -0.214 to 0.302, P = 0.686).@*CONCLUSION@#The posterior tibial slope between 3° and < 8° can be recommended to improve knee joint function in mobile UKA, and excessive or insufficient PTS should be avoided.

Screening specific minimum amino acid sequence triggering immunity to enterovirus 71 / 中华微生物学和免疫学杂志

Objective:To screen the neutralizing epitope of enterovirus 71 (EV71) and determine the specific minimum amino acid sequence that triggers immunity for providing a theoretical basis for the development of synthetic peptide vaccines.Methods:EV71 neutralizing antibody-specific binding clones were panned and sequenced using a phage display random 12-peptide library to obtain the key sequences of neutralizing epitopes. A series of peptides containing the key sequences with N-terminal acetylation (AC) and C-terminal linking to Keyhole limpet hemocyanin (KLH) were synthesized. Serum samples were collected after immunizing mice with the modified peptides. Then the immunogenicity of the peptides and the neutralizing activity of serum samples were analyzed by Western blot, ELISA and neutralization test.Results:After three rounds of panning, cloning and sequencing, KQEKDL was identified as the key motif. The serum samples collected from the mice immunized with the modified series of peptides containing key motifs had different degrees of binding ability to EV71 and VP1 protein. The serum samples of mice immunized the synthetic peptide containing only the minimum key motif (AC-KQEKDL-KLH) had the strongest response to the other three peptides and EV71 and the highest neutralizing titer.Conclusions:The EV71 neutralizing epitope was successfully screened using the phage display random peptide library. The key motif of KQEKDL might be the specific minimum amino acid sequence that triggered the immune system. This study provides a theoretical basis for better understanding the immune response mechanism, evaluating the immunogenicity of the antigens and further research and development of polypeptide vaccines.

Effects of live attenuated polio vaccine on the immunogenicity of live attenuated rotavirus vaccine / 中华微生物学和免疫学杂志

Objective To evaluate whether simultaneous vaccination with live attenuated polio vaccine affects the immunogenicity of live attenuated rotavirus ( RV) vaccine. Methods Rotarix produced by GlaxoSmithKline was used as the research object. Two doses of Rotarix were orally administered on day 0 and month 1, and oral live attenuated polio vaccine (OPV) was administered on day 0, month 1 and month 2 according to the national vaccination plan. Healthy infants aged 6 to 16 weeks were randomly divided into two groups:interval vaccination group ( Rotarix and OPV were vaccinated on different days) and simultane-ous vaccination group ( Rotarix and OPV were vaccinated on the same day) . Serum samples were collected on day 0, month 2 and month 12, and serum RV-IgA was measured by enzyme linked immunosorbent assay. Statistical analysis was performed to evaluate whether there were statistical differences in the seroconversion rate and level distribution of RV-IgA between the two groups. Results The seroconversion rate of serum RV-IgA in month 2 was 73. 84％ in the interval vaccination and 63. 95％ in the simultaneous vaccination group, and the difference between them was statistically significant (P<0. 05). The geometric mean concen-trations (GMC) of RV-IgA were 97 EU/ml and 90 EU/ml, respectively (P>0. 05). Compared with the simultaneous vaccination group, the seroconversion rate and GMC of serum RV-IgA in month 12 were higher in the interval vaccination group, and the differences were statistically significant (P<0. 05). Conclusions Simultaneous vaccination with live attenuated polio vaccine would affect the immune response of live attenua-ted rotavirus vaccine, especially the maintenance of RV-IgA antibody level.

Effects of live attenuated polio vaccine on the immunogenicity of live attenuated rotavirus vaccine / 中华微生物学和免疫学杂志

Objective@#To evaluate whether simultaneous vaccination with live attenuated polio vaccine affects the immunogenicity of live attenuated rotavirus (RV) vaccine.@*Methods@#Rotarix produced by GlaxoSmithKline was used as the research object. Two doses of Rotarix were orally administered on day 0 and month 1, and oral live attenuated polio vaccine (OPV) was administered on day 0, month 1 and month 2 according to the national vaccination plan. Healthy infants aged 6 to 16 weeks were randomly divided into two groups: interval vaccination group (Rotarix and OPV were vaccinated on different days) and simultaneous vaccination group (Rotarix and OPV were vaccinated on the same day). Serum samples were collected on day 0, month 2 and month 12, and serum RV-IgA was measured by enzyme linked immunosorbent assay. Statistical analysis was performed to evaluate whether there were statistical differences in the seroconversion rate and level distribution of RV-IgA between the two groups.@*Results@#The seroconversion rate of serum RV-IgA in month 2 was 73.84% in the interval vaccination and 63.95% in the simultaneous vaccination group, and the difference between them was statistically significant (P<0.05). The geometric mean concentrations (GMC) of RV-IgA were 97 EU/ml and 90 EU/ml, respectively (P>0.05). Compared with the simultaneous vaccination group, the seroconversion rate and GMC of serum RV-IgA in month 12 were higher in the interval vaccination group, and the differences were statistically significant (P<0.05).@*Conclusions@#Simultaneous vaccination with live attenuated polio vaccine would affect the immune response of live attenuated rotavirus vaccine, especially the maintenance of RV-IgA antibody level.

Validation of a cell infection-based quantitative RT-PCR for evaluation of rotavirus vaccine potency / 中华微生物学和免疫学杂志

Objective To validate a cell infection-based quantitative RT-PCR for evaluating the potency of rotavirus vaccine. Methods According to the ICH ( the International Council for Harmonization) Harmonised Tripartite Guideline, the method was validated for its specificity, accuracy, precision, linearity and robustness. Results The method had good specificity as it could only amplify and detect the corre-sponding type of rotavirus strain. The recovery rates for determining the potency against rotaviruses of G2, G3 and G4 types were 97％ to 108％. The percent coefficient of variation ( CV) of both intra-plate and in-ter-plate precision was≤2. 62％, while the intraday and interday CV was≤1. 76％ and≤2. 27％, respec-tively. The CV between the two experimenters was≤7. 68％. The linearity range of the method was 4. 4-6. 5 UI for G2 type rotavirus, 3. 9-8. 3 UI for G3 type and 3. 5-8. 1 UI for G4 type. Good robustness was observed using the cells of 140 to 160 generations. Conclusions The cell infection-based quantitative RT-PCR was shown to have satisfactory specificity, accuracy, precision, linearity and robustness, suggesting that it was a suitable method for evaluating the potency of multivalent rotavirus live vaccines.

The protective effects of lycium barbarum polysaccharides on retinal neurons in diabetic rats and its mechanism / 中国应用生理学杂志

OBJECTIVE@#To clarify whether lycium barbarum polysaccharides (LBP) have protective effects on retina neuronal cells in diabetic rats and to identify the related mechanism involved in this process.@*METHODS@#Eighteen SD rats were randomly divided into 3 groups ( n= 6): normal control group (NC), diabetes mellitus group (DM) and LBP-treatment group (DM+LBP). The diabetic rat model was induced by single intraperitoneal injection of streptozotocin (STZ). The rats in DM+LBP group were treated with LBP at the dose of 1 mg/kg by gavage, once a day for 12 weeks. After the treatment, the weight and blood glucose, the generation of reactive oxygen species (ROS), the surviving retinal ganglion cells (RGCs) and amacrine cells and the protein expressions of nuclear factor E2-related factor 2 (Nrf2) and the heme oxygenase-1 (HO-1) were detected.@*RESULTS@#The successful rate of diabetic model was 100%. Compared with NC group, the rats of DM group caused weight loss, elevated blood glucose, a marked increase of ROS generation and a significant decrease in the number of RGCs and amacrine cells (P＜0.01), and these effects were diminished or abolished by LBP treatment. Meanwhile, LBP significantly increased the expressions of Nrf2 and HO-1 in the retinas of diabetic rats (P＜0.01).@*CONCLUSION@#LBP can improve retinal oxidative stress and exert beneficial neuroprotective effects in diabetic rats, and its mechanism may be associated with the activation of the Nrf2/HO-1 antioxidant pathway.

Progress in developing Norovirus vaccines based on virus-like particles / 中华微生物学和免疫学杂志

Human norovirus (NoV) is the major cause of human acute non-bacterial gastroenteritis worldwide. The development of NoV vaccines is limited by the inability to culture cells in vitro and the lack of small animal models. Thus, traditional methods used to prepare live attenuated vaccines are not suitable for preparing NoV vaccines. Subunit vaccines against NoV infection, especially virus-like particle (VLP)-based vaccines, have outstanding advantages in the research and development of NoV vaccines. In this review, we focused on reviewing recent advances in the fields of NoV VLP-based vaccines and the development of NoV VLP-based vaccines using different expression systems as well as identifying the research direction for NoV VLP-based vaccines.

Norovirus P particle and its application / 中华微生物学和免疫学杂志

The challenges posed by norovirus infections to global health are increasing accompa-nied by the rapid rate of the genetic and antigenic evolution of circulating noroviruses. Due to lack of in vitro culture cells and small animal models, norovirus vaccine cannot be prepared by using traditional techniques. With the in-depth understanding and study of norovirus, the subunit vaccines against norovirus infection based on P particles have been developed and presented the characteristics of easily expressed, low cost, high immunogenicity, stable structure and so on. In addition, norovirus P particle has been used as a subvi-ral nanoparticle for vaccine development against other viruses and for antibody production against chronic dis-ease ( Alzheimer′s disease) , which benefits from the accommodation of foreign antigens in the three loops of P particle. In this review, we describe the progresses in the field of P particle related vaccines for providing suggestions about the research and development of multivalent vaccines in China.

Application of TG-ROC analytical method in detection reagent quality evaluation / 国际检验医学杂志

Objective To compare the relationship between the enzyme‐linked immunosorbent assay(ELISA) reagent and West‐ern blot(WB) confirmation reagent for analyzing the quality lever of human T‐cell lymphotropic virus(HTLV) detection reagent . Methods The WB confirmation reagent was used to detect anti‐HTLV antibody in 156 human serum samples of ELISA prelimina‐ry screening positive .The ELISA cut‐off value(optimal value) was selected by using the two‐graph receiver operating characteristics (TG‐ROC) analytical method .The two‐by‐two table analysis was constructed to analyze the consistency of results detected by the two methods ,moreover the McNemar test was used to evaluate the consistency of detection results .The quality level of HTLV de‐tection reagent was comprehensively evaluated .Results Among 156 serum samples of ELISA preliminary screening positive ,only 40 samples were positive by the WB confirmation ,and other 116 samples were negative .The sensitivity and specificity of ELISA de‐tection reagent obtained by TG‐ROC analysis were 97 .5% and 45 .7% respectively ,the TG‐ROC test also indicated that the detec‐tion results had significant difference between ELISA and WB(P<0 .05) .By adjusting the cut‐off value ,the sensitivity and specific‐ity of ELISA were increased to 88 .8% (parametric method) .In the comparison of the parametric method and the non‐parametric method ,the obtained areas under the curve(AUC) was 0 .923 5(parametric method) ,their results were basically consistent .Conclu‐sion Although above results indicate that the detection results of ELISA reagent are different from those of WB ,but adjusting the cut off value can increase its sensitivity and specificity ,thus increases the reliability of diagnosis result .

The effects of lymph after intestinal ischemia/reperfusion on the inflammatory factors and Toll-like receptor 4 ligand high mobility group box-1 in Toll-like receptor 4 deficient mice / 中华临床营养杂志

Objective To investigate the effects of lymph from ischemic/reperfused intestine on the inflammatory factors and Toll-like receptor 4 (TLR4) ligand high mobility group box-1 (HMGB1) in TLR4 deficient (TLR4-/-) mice.Methods A total of 20 SD rats weighing (300 ±20) g were randomly assigned into two groups:lymph drainage group (group N,lymph drainage for 180 minutes without other treatment) and intestinal ischemia/reperfusion group (group I/R,draining the lymph for 180 minutes while clipping the superiormesenteric artery for 60 minutes followed by 120-minute reperfusion).Thirty-two TLR4-/-mice and thirty-two C57BL/6 wild type (WT) mice were each divided into 4 sub-groups (n =8),injected with different fluids through the caudal vein:group N with normal lymph;group I/R with I/R lymph;group Edt with endotoxin;group HMGB1 with HMGB1 protein.The mice were sacrificed 180 minutes after the injection for sample collection.Results The levels of endotoxin and HMGB1 in the lymph drainage of the group I/R rats were significantly higher than that of the group N rats [(0.034 ± 0.050) Eu/ml vs.(0.017 ± 0.023) Eu/ml,P =0.033;(4.293 ± 0.883) ng/ml vs.(0.509 ± 0.128) ng/ml,P =0.006].In the mice injected with HMGB1,the mucosa thickness and villus height in the ileum of the WT mice were significantly lower than that of the TLR4-/-mice [(335.8±43.2) μmvs.(602.1±37.5) μm,P=0.000;(273.0±31.7) μm vs.(404.5 ± 18.6) μm,P =0.000];in both WT and TLR4-/-mice injected with the I/R lymph drainage,the mucosa thickness and virus height were decreased,but the decrements were significantly lower in TLR4-/-mice;there were no statistically significant differences in the levels of interleukin-6 (IL-6),tumor necrosis factor-α (TNF-α),endotoxin,and HMGB1 between the TLR4-/-and the WT mice injected with normal lymph or endotoxin.In the mice injected with I/R lymph drainage,the levels of inflammatory factors in the TLR4-/-mice were significantly lower than those in the WT mice [TNF-α:(28.637 ±5.166) pg/ml vs.(41.917 ±8.175) pg/ml,P=0.000;IL-6:(60.900 ±24.729) pg/ml vs.(110.265 ±28.545) pg/ml,P =0.000].In the mice injected with HMGB1,the levels of inflammatory factors in the TLR4-/-mice were significantly decreased compared with those in the WT mice [TNF-α:(20.865 ± 6.464) pg/ml vs.(31.059 ± 6.204) pg/ml,P=0.004;IL-6:(36.268 ±8.977) pg/ml vs.(76.677 ± 14.099) pg/ml,P=0.000].Conclusions The concentrations of endotoxin and HMGB1 are significantly increased during intestinal I/R in rats.After injection of I/R lymph drainage,endotoxin,and HMGB1,the levels of inflammatory factors and HMGB1 in the mice injected with I/R lymph drainage are significantly higher than those in the mice injected with normal lymph;the levels of inflammatory factors and local damage of intestinal mucosa are significantly reduced in the TLR4-/-mice than in the WT mice.The gut-lymph pathway may play a key role in the intestinal I/R injury.

Simultaneous determination of danshensu, hydroxysafflor yellow A, rosmarinic acid, lithospermic acid, salvianolic acid B in water extract of mixed salviae miltiorrhizae radix et rhizoma and carthami flos by HPLC / 中国中药杂志

<p><b>OBJECTIVE</b>To develop an HPLC method to determine the contents of danshensu, hydroxysafflor yellow A, rosmarinic acid, lithospermic acid, salvianolic acid B in the water extract of mixed Salviae Miltiorrhizae Radix et Rhizoma and Carthami Flos simultaneously.</p><p><b>METHOD</b>The separation were carried out at 30 degrees C on a ZORBAX Eclipse Plus C18 column (4.6 mm x 100 mm, 1.8 microm) with formic acid-500 mmol x L(-1) ammonium formate-water solution (0.5:10:90) as mobile phase A and acetonitrile-formic acid solution (100: 0.5) as mobile phase B in gradient mode at a flow rate of 0.5 mL x min(-1). Detection wavelengths were 280 nm for danshensu, rosmarinic acid, lithospermic acid, salvianolic acid B, and 380 nm for hydroxysafflor yellow A.</p><p><b>RESULT</b>The 5 components were separated well with a good linearity (R2 > 0.999 3) in the range of the test concentration. The average recoveries of danshensu, hydroxysafflor yellow A, rosmarinic acid, lithospermic acid, and salvianolic acid B were 99.1%, 102%, 102%, 98.5% and 101%, respectively.</p><p><b>CONCLUSION</b>This method is simple, accurate, and repeatable.</p>

Feedforward control strategy and its application in quality improvement of ethanol precipitation process of danhong injection / 中国中药杂志

In this work, a feedforward control strategy basing on the concept of quality by design was established for the manufacturing process of traditional Chinese medicine to reduce the impact of the quality variation of raw materials on drug. In the research, the ethanol precipitation process of Danhong injection was taken as an application case of the method established. Box-Behnken design of experiments was conducted. Mathematical models relating the attributes of the concentrate, the process parameters and the quality of the supernatants produced were established. Then an optimization model for calculating the best process parameters basing on the attributes of the concentrate was built. The quality of the supernatants produced by ethanol precipitation with optimized and non-optimized process parameters were compared. The results showed that using the feedforward control strategy for process parameters optimization can control the quality of the supernatants effectively. The feedforward control strategy proposed can enhance the batch-to-batch consistency of the supernatants produced by ethanol precipitation.

An approach to determine critical process parameters for ethanol precipitation process of danhong injection / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a method integrating multi-targets for determining critical process parameters of the manufacturing process of traditional Chinese medicine.</p><p><b>METHOD</b>The ethanol precipitation process of Danhong injection was taken as an application case of the method established. Fractional factorial design of experiments were conducted. Mathematical models relating seven process parameters to ten targets in the ethanol precipitation process were established. Then the sums of the absolute values of the regression coefficients in the models were used to evaluate the criticality of process parameters.</p><p><b>RESULT</b>Water content in the concentrate, ethanol concentration and ethanol consumption were identified as the critical process parameters.</p><p><b>CONCLUSION</b>The method established can integrate multi-targets effectively for the evaluation of critical process parameters.</p>

Application of ultraviolet spectroscopy for rapid analysis in extraction process of danhong injection / 中国中药杂志

In this work, a rapid analysis method basing on ultraviolet spectroscopy was established for the determination of danshensu, protocatechuic aldehyde, rosmarinci acid, lithospermic acid and salvianolic acid B in the extraction process of Danhong injection. In the extraction process of Danshen and Honghua crude drugs, 44 extraction solution samples were collected and the contents of the five components were determined by HPLC analysis. The ultraviolet spectra of the samples were collected. Partial least square regression was used to establish the multivariate calibration models between the ultraviolet spectra and the contents of the five components. The results showed that the established models could predict the contents of the five components in the extraction solution accurately. The ultraviolet spectroscopy method established in this work can be used for rapid analysis of the intermediates of Danhong injection, which may be applied for the quality control in the manufacturing process.

Histopathology research of Cinnamaldehyde as pulp-cap of pulpotomy in rats / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the stimulation of Cinnamaldehyde to the pulp tissue and the periapical tissue of rats' teeth, to provide evidence for developing Cinnamaldehyde as a pulp-cap of pulpotomy in primary teeth.</p><p><b>METHODS</b>Using Cinnamaldehyde as pulp-cap in pulpotomy of rats' teeth, set up Cinnamaldehyde group, formaldehyde cresol formocresol group and blank group. After different treatment according to the empirical procedure, rats were killed in the 4th week and on the 12th week. Then the experiment teeth and the periodontal tissue were made into HE slides and observed using light microscope.</p><p><b>RESULTS</b>The 4" week, internal absorption, external absorption, and calcification were of no significance among all the groups. Inflammation in blank group was far more severe than that in other two groups (P<0.05), while the latter two were of no significance. The 12th week, internal absorption, external absorption, and inflammation between the former two groups were of no differences, but the indexes were significantly different from them of the blank group (P<0.005). Calcify was not obvious in all the slides.</p><p><b>CONCLUSION</b>As a pulp-cap, Cinnamaldehyde stimulates the pulp tissue and the periapical tissue at a very low level. The research provides histopathology rationale for Cinnamaldehyde as pulp-cap of pulpotomy in primary teeth.</p>

Quantitative study of effects of cinnamaldehyde on levels of endotoxin in root canals in rats' periapical periodontitis model / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To provide experimental evidence for the exploitation of cinnamaldehyde as a kind of root canal disinfectant through studying the effect of cinnamaldehyde on endotoxin in root canals.</p><p><b>METHODS</b>This experimental model of periapical periodontitis was established with Wistar rats. The 75 rats were divided randomly into 3 groups: Group of cinnamaldehyde, group of formaldehyde cresol formocresol, group of physiological saline. The level of endotoxin was measured by quantitative chromogenic tachypleus amebocyte lysate method before and after sealing the drugs in the root canal.</p><p><b>RESULTS</b>The level of endotoxin in the group of cinnamaldehyde and formaldehyde cresol formocresol decreased obviously (P < 0.05), and the difference between them was of no significance (P > 0.05), the group of physiological saline was of no significant difference (P > 0.05).</p><p><b>CONCLUSION</b>Cinnamaldehyde can decrease the level of endotoxin obviously.</p>

Screening and characterization of DNA aptamers with hTNF-alpha binding and neutralizing activity / 生物工程学报

Human tumor necrosis factor alpha (hTNF-alpha) is one of the most important inflammatory cytokines that acts as a mediator in inflammatory and immune response and plays a key role in host defense against infection. The over expression of hTNF-alpha is associated with serious consequences, such as shock, hypotension, thrombus, septicemia and even death. It has been implicated in many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, chronic heart failure and septic shock. Inhibiting the bio-activity of hTNF-alpha is one of the strategy for the treatment of these diseases. Compared with traditional recombinant protein drugs, small molecule drugs have many advantages, such as high affinity, low immunogenecity and low cost. Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the selection of oligonucleotides that bind with high affinity and specificity to target proteins. Such oligonucleotides are called aptamers, and are potential therapeutics for blocking the activity of pathologically relevant proteins. To obtain oligonucleotide aptamers specifically binding to TNF, a 40nt random DNA combinatorial library flanked by 31nt fixed sequences was chemically synthesized. The random library was amplified with PCR and subjected to selection by SELEX protocol against hTNFalpha. After incubation of the library with hTNFalpha, the mixture was blotted onto Immobilon-NC transfer membrane. The no-specific binding was washed away and the hTNFa binding aptamers were eluted and detached from the target protein. The eluted oligo nucleotides were amplified with PCR and served as the DNA library for the next round selection. After 12 rounds of such selection, the selected aptamers were cloned to pGEM-T vector. Positive clones were identified by restriction enzyme digestion and DNA sequencing. Oligo DNA were synthesized according to the sequence data and tested for their activities. Binding activity of the aptamers to hTNFalpha were detected by ELISA and dot blot with biotin-streptavidin-horseradish peroxidase system. Mouse L929 cells were used to test the anti-hTNFa activity of the DNA aptamers. The aptamers were incubated with hTNFalpha and added to the L929 cells. The results were read under microscope and with MTT staining. It was shown that these DNA aptamers bound to hTNFalpha with high affinity, and can inhibit the cytotoxicity of hTNFalpha on cell culture. The affinity of these aptamers are different and may related to their structure. These ssDNA aptamers are potential for the treatment and diagnosis of hTNFalpha related diseases.