[Impact of autologous hematopoietic stem cell transplantation on the efficacy of CAR-T treatment of relapsed/refractory multiple myeloma].

Objective: To evaluate the effect of autologous hematopoietic stem cell transplantation (ASCT) on the treatment of relapsed/refractory multiple myeloma (RRMM) with chimeric antigen receptor T cell (CAR-T) therapy. Methods: A retrospective cohort study. The clinical data of 168 patients with RRMM who underwent CAR-T therapy at the Department of Hematology, Xuzhou Medical University Hospital from 3 January 2020 to 13 September 2022 were analyzed. Patients were classified into a transplantation group (TG; n=47) and non-transplantation group (NTG; n=121) based on whether or not they had undergone ASCT previously. The objective response rate (ORR), progression-free survival (PFS), overall survival (OS) and the levels of CD3, CD4, CD8, CD19, CD56 and natural killer (NK) cells before CAR-T infusion were analyzed by χ2 test, Kaplan-Meier method and independent sample t-test. Results: Among 168 patients with RRMM, 98 (58.3%) were male. The median age of onset was 57 (range 30-70) years. After CAR-T therapy, the ORR of patients was 89.3% (92/103) in the NTG and 72.9% (27/73) in the TG. The ORR of the NTG was better than that of the TG (χ2=5.71, P=0.017). After 1 year of CAR-T therapy, the ORR of the NTG was 78.1% (75/96), and that of the TG was 59.4% (19/32). The ORR of the NTG was better than that of the TG (χ2=4.32, P=0.038). The median OS and PFS in the NTG were significantly longer than those in the TG (OS, 30 vs. 20 months; PFS, 26 vs. 12 months; both P<0.05). The CD4 level before CAR-T infusion in the TG was significantly lower than that in the NTG (25.65±13.56 vs. 32.64±17.21; t=-2.15, P=0.034), and there were no significant differences in the counts of CD3, CD8, CD19, CD56, and NK cells between the TG and NTG (all P>0.05). Conclusion: Among patients suffering from RRMM who received CAR-T therapy, patients who did not receive ASCT had significantly better outcomes than those who had received ASCT previously, which may have been related to the CD4 level before receiving CAR-T therapy.

Trauma to erythrocytes induced by long term in vitro pumping using a roller pump.

The objective of this study is to investigate the impact of trauma on erythrocyte caused by long term in vitro pumping using roller pump. Ten bags of human blood (400 ml each) were provided by a local blood bank and they were divided into two groups with five bags in each group. Each blood bag was subject to pumping in a closed circuit, which was composed of silica gel tubes and a roller pump. Polystan and COBE pumps were used for the two groups, respectively. The blood was pumped for 16 h in vitro. Free hemoglobin (FHb), platelets (PLT), erythrocyte fragility (EF), and morphological analysis of erythrocytes observed under scanning electron microscope were measured to evaluate the impact of trauma on erythrocytes. A small amount of blood was collected for analysis before pumping, at the end of the 4th hour and then every 2 h till the end of the 16th hour. Some blood samples were also collected for electron microscope scanning before pumping and every 4 h during pumping. It was found that FHb and PLT linearly increased with the pumping time. There was a significant correlation between the two parameters (r=0.7745, p<0.001). The hemolysis indexes of the two groups were 0.296 and 0.3993 mg/L/h, respectively, with no significant difference. During the pumping process, EF changed slightly. The observation of scanning electron microscopy showed various deformed erythrocytes after pumping, including the distortion of cell membrane and the appearance of echinocytes, which increased with pumping time. This study demonstrated that long term pumping using roller pump not only caused the immediate rupture of red blood cells, i.e. the immediate hemolysis, but also caused sub-trauma to a large number of erythrocytes, which led to the delayed hemolysis. The change of erythrocyte morphology was the basis of the delayed hemolysis.

Rapid detection of Mycobacterium tuberculosis in various clinical specimens by using polymerase chain reaction combined with a nonradioactive hybridization system.

A DNA amplification system using the polymerase chain reaction (PCR) combined with a nonradioactive digoxigenin-labeled probe hybridization was employed to detect Mycobacterium tuberculosis in clinical specimens. One hundred and thirty specimens were tested by several methods including routine culture method, acid-fast staining, BACTEC 460 detection system, PCR, and PCR-hybridization techniques. Sixteen out of 130 specimens were culture positive on Middlebrook 7H11 agar, 10 were positive with acid-fast staining, 18 were positive with BACTEC 460 detection system, 23 were positive with PCR technique, and 62 were positive with PCR-nonradioactive hybridization technique. When compared with culture results, PCR-nonradioactive hybridization had an overall sensitivity of 100% (16/16) and a specificity of 59.7% (68/114). However, 28 out of 46 (60.9%) PCR-nonradioactive hybridization positive specimens which were culture negative had clinical data supporting the diagnosis of tuberculosis. In addition, 4 specimens which were negative by routine culture but positive by BACTEC 460 detection system and two specimens which were negative by routine culture but positive by acid-fast staining were all positive by PCR-hybridization technique. These data suggest that routine culture method may not be sensitive enough to detect M. tuberculosis in all kinds of clinical specimens. Taking this deviation into account, the specificity of PCR-nonradioactive hybridization technique may be rectified range from 63% (68/108) to 79.1% (68/86). PCR itself is not satisfactory enough to detect M. tuberculosis in specimens (the sensitivity and specificity were 56.3% and 87.7%, respectively) in this study. However, when it combines with DNA hybridization technique, they can be a very powerful and rapid diagnostic tool to detect M. tuberculosis in clinical specimens.

Molecular cloning and expression of the 01 rfb region from a pyelonephritic Escherichia coli 01:H1:K7.

The genes responsible for the biosynthesis of the O1 polysaccharide from a human pyelonephritic Escherichia coli were cloned and expressed in a rfb-deleted E. coli K-12 strain. Deletion analysis of the clone demonstrated that a DNA fragment size larger than 6.7 kb and smaller than 10 kb is responsible for O1-antigen biosynthesis.

A previously undescribed plasmid and African R plasmid in Neisseria gonorrhoeae isolated in Taiwan.

A new undescribed plasmid, 15.6-Mdal in size, was detected in Neisseria gonorrhoeae isolates in Taiwan. The plasmid was co-existent with 2.6-Mdal and 7.8-Mdal plasmids in three out of 190 clinical isolates. It appeared to be consisted of six copies of the 2.6-Mdal plasmids as evidenced by restriction endonuclease cleavage. In addition, African 3.2-Mdal R plasmids have also been detected in penicillinase-producing N. gonorrhoeae (PPNG) isolates in Taiwan. They accounted for 7% of PPNG.

Serogrouping, plasmid and drug resistance of Pseudomonas aeruginosa in Taiwan.

The Pseudomonas aeruginosa isolates in Taiwan were grouped with antisera against O antigens and tested with 13 different antimicrobial agents. Group E (28%) lead in serogrouping, followed by Group G (19%), Group F (16%), Group B and Group H (12%, respectively), and there were no significant differences in distribution of serogroups among the isolates from different areas in Taiwan. Amikacin (75%) and piperacillin (74%) were the most effective antibiotics, followed by tobramycin (63%), carbenicillin (62%), ticarcillin (57%) and gentamicin (56%). Group F strains were relatively more susceptible to most of the antibiotics tested than strains of other serogroups. All strains tested were multiple drug resistance. Thirty percent (67/224) of P. aeruginosa carried plasmids. The average number of resistant markers per strain was 8.7 for plasmid-carrying strains as compared to 7.6 for plasmid-free strains. The plasmid-carrying strains showed higher drug resistance than plasmid-free strains. Twenty-seven percent (13/48) of plasmid-carrying strains contained R plasmids which were detected by transformation (6/23) and conjugation (7/25). These R plasmids totally encoded at least seven different resistant markers. The R plasmid from P. aeruginosa 119 was not modified after transforming into Escherichia coli RR1. The R plasmids of P. aeruginosa transferred into E. coli RR1 or Serratia marcescens 73 caused some changes in the outer membrane of transformants or transconjugants. Some R plasmid-carrying strains and their transformants or transconjugants exhibited the ability to produce acetyltransferase.

Auxotyping of Neisseria gonorrhoeae isolated in Taiwan.

One hundred and seventy eight out of 319 clinical isolates (55.8%) of Neisseria gonorrhoeae isolated in Taiwan were identified as penicillinase-producing Neisseria gonorrhoeae. Two hundred and ninety six isolates were subdivided into 24 distinct auxotypes. The majority of gonococci tested were non-requiring (43.6%) and proline-dependent (43.2%). No arginine-hypoxanthine-uracil requiring strain was obtained in this study. Only one arginine-requiring strain was obtained from 296 isolates. Different auxotypes of gonococci were isolated from the same patient who had contracted gonorrhea. Each of 17 patients was infected by at least two different auxotypes of gonococci.

Drug resistance, R plasmids and pigmentation of Serratia marcescens isolated in Taiwan.

The Serratia marcescens isolates used in this study were resistant to ampicillin, tetracycline or cephalothin, streptomycin, tobramycin, kanamycin, carbenicillin, chloramphenicol and gentamicin in descending order. Nalidixic acid was the most effective antibiotic against S. marcescens, followed by amikacin and sulfamethoxazole-trimethoprim. The non-pigmented plasmid-carrying isolates displayed higher resistance to some antimicrobial agents than did the pigmented isolates and plasmid-free white isolates. Nine out of 12 resistant markers were coded by plasmids in S. marcescens. The average number of resistant markers per strain was seven for plasmid-containing white isolates as compared to four for other S. marcescens groups. About 73% of S. marcescens contained plasmids. Thirty eight percent of plasmid-carrying S. marcescens spread their R plasmids to E. coli. Conjugative R plasmids were identified in six out of 17 strains of S. marcescens, which apparently contained a single plasmid.

Isolation and characterization of Yersinia enterocolitica in Taipei.

Of 187 food samples analyzed, 3(1.6%) displayed the presence of Yersinia enterocolitica. All of them were isolated from frozen food samples. Two strains were isolated from 21 sliced beef samples (9.5%), and were identified as serotype O5. One strain was isolated from 20 egg dumpling samples (5%), and was typed as serotype O9. No Y. enterocolitica was found in refrigerated food samples. In addition to serotypes O5 and O9, serotypes O3 and O8 were also identified for other local strains that were included in this study. Three different biotypes have been determined for these strains according to Wauter's schema. Most of the strains tested for their antimicrobial susceptibility were resistance to carbenicillin, ampicillin and cephalothin, and all were susceptible to aminoglycosides, tetracycline, nalidixic acid, chloramphenicol, and sulfamethoxazole-trimethoprim. Five out of 7 strains carried plasmids, but these plasmids may not code for drug resistance.

Protective effect of normal guinea pig serum on the bactericidal activity of normal human serum for Neisseria gonorrhoeae.

Normal human serum (NHS) killed Neisseria gonorrhoeae (GC) strain F62 after incubation at 37 degrees C. In contrast, the bactericidal effect of normal guinea pig serum (NGPS) under similar conditions was not obvious. When NHS was mixed with NGPS or heated NGPS and a suspension of strain F62, the bacteria were protected from killing by NHS. When strain F62 was incubated first with NGPS for 30 min at 37 degrees C, washed and then treated with NHS, the strain was significantly protected. Depletion of complement (C) of NGPS by zymosan treatment or by heating at 56 degrees C for 30 min decreased the protective activity of NGPS. These data suggest that protection of GC by NGPS occurred on the surface of GC and C may be involved. However, when strain F62 was incubated first with heated NGPS for 30 min at 37 degrees C, washed and then treated with NHS, the strain was significantly killed. This suggests that protection of GC by heated NGPS did not occur on the surface of GC. In addition to the protective effect of NGPS on the bactericidal activity of NHS for GC, NGPS adsorbed with strain F62 also had the ability to kill the GC.

Enzyme immunoassay for detection of Neisseria gonorrhoeae antigens.

An enzyme immunoassay (EIA; Gonozyme, Abbott Laboratories) for the detection of Neisseria gonorrhoeae antigens was assessed. Clinical isolates of N. gonorrhoeae or specimens obtained from male urethra or female cervix were tested by EIA. EIA results were compared with results of conventional culture identification for N. gonorrhoeae. Fifty clinical isolates of gonococci reacted positively with Gonozyme. Five strains of E. coli showed negative reaction to Gonozyme. Five strains of nonpathogenic Neisseria reacted differently with Gonozyme depending upon the viable number of the test organisms. The sensitivity and specificity of EIA for specimens form both sex were 72.4% and 93.8%, respectively. The corresponding figures for the male specimens were 84.6% and 71.4%, and for the female specimens 62.5% and 95%, respectively.

Induced changes in the surface of Neisseria gonorrhoeae.

Growth of Neisseria gonorrhoeae strain F62 on medium containing pyruvate and a high ratio of cysteine to cystine resulted in functional and structural changes that are consistent with phenotypic changes in lipopolysaccharide. Both transparent (O-) and moderately opaque (O+) variants became more sensitive to killing by normal human serum and resistant to killing by pyocin G, a bacteriocin from Pseudomonas aeruginosa. Electrophoresis of outer membranes in the presence of sodium dodecyl sulfate demonstrated differences also dependent upon the growth medium. When gels were treated with periodic acid and stained with silver, lanes containing outer membranes obtained after growth in the modified medium demonstrated two bands in addition to those independent of the growth medium. The enhancement of these additional bands by periodate treatment indicated that they represent material containing carbohydrate. The mechanism by which the changes in the growth medium affected the surface of N. gonorrhoeae is not known; however, the changes demonstrated by electrophoresis were dependent upon either the high concentration of cysteine or the high ratio of cysteine to cystine.

Biosynthesis of prodigiosin by white strains of Serratia marcescens isolated from patients.

Serratia marcescens isolated from infected adults generally does not synthesize prodigiosin. Other investigators have reported that most clinical strains form a pigment if furnished with 4-methoxy-2,2'-bipyrrole-5-carboxyaldehyde (MBC), a precursor of prodigiosin. To determine whether the pigment was prodigiosin, we studied 65 white strains of S. marcescens isolated from patients. On the basis of response to MBC, we assigned the strains to one of three classes: class 1 (14 strains), strains remaining white; class 2 (48 strains), strains becoming gray or pink; and class 3 (3 strains), strains becoming blue. Ethanol extracts of bacteria of classes 2 and 3 did not behave like prodigiosin when acidified or alkalinized, and the pigment spectra were not similar to prodigiosin spectra. If strains of class 3 were furnished with MBC plus 2-methyl-3-amylpyrrole (MAP), the other immediate precursor of prodigiosin, the pigment synthesized was characteristic of prodigiosin. Strains of classes 1 and 2 responded identically to MBC plus MAP and MBC alone. Although the majority of S. marcescens white strains from patients formed pigments in the presence of MBC, the pigments were not prodigiosin. A few strains did synthesize prodigiosin, but only if furnished with both MBC and MAP.

Pyocyanine production by Pseudomonas aeruginosa.

Dextrose enhanced the growth of P. aeruginosa but suppressed the biosynthesis of pyocyanine. The preformed pigment could be released from dead cells. Pigmentation was not correlated directly with number of viable organisms in the culture. High concentration of maltose likewise inhibited pyocyanine production. Maltose contained in medium used for pyocyanine production by P. aeruginosa should be kept in low concentration or omitted.