Regional Injection of CAR-T Cells for the Treatment of Refractory and Recurrent Diffuse Large B Cell Lymphoma: A Case Report.

BACKGROUND: Lymphoma is a common hematological malignancy with many subtypes and considerable heterogeneity. Traditional treatments include chemotherapy, radiotherapy, and surgery. Patients with relapsed, refractory or advanced stage lymphoma have a dismal prognosis. In recent years, chimeric antigen receptors (CARs) have been recognized as powerful tools that redirect antigen-specific T cells independent of human lymphocyte antigen (HLA) restriction and specifically kill tumor cells. Satisfactory results with CAR-based treatments have been achieved in relapsed/refractory B cell leukemia/lymphoma. Our center explored the strategy of subcutaneous injections combined with intravenous drip to overcome certain issues. CASE PRESENTATION: A patient with stage IV refractory and relapsed diffuse large B cell lymphoma was treated with regional and intravenous CAR-T cells. During the observation period, the temperature of the skin at the abdominal wall mass was slightly elevated, and tolerable pain in the injection area was reported. Imaging showed regional liquefactive necrosis. After the sequential administration of ibrutinib and venetoclax, the abdominal wall mass significantly decreased in size. CONCLUSION: The regional injection of CAR-T cells might be safe and feasible for the treatment of regional lesions in patients with refractory and relapsed advanced lymphoma.

Expression of key ion channels in the rat cardiac conduction system by laser capture microdissection and quantitative real-time PCR.

The objective of this study was to investigate the molecular basis of the inferior nodal extension (INE) in the atrioventricular junctional area that accounts for arrhythmias. The INE was separated from the adult rat heart by laser capture microdissection. The mRNA expression of ion channels was detected by quantitative real-time PCR. Hierarchical clustering was used to demonstrate clustering of expression of genes in sections. The mRNA expression of HCN4, Ca(v)3.1 and Ca(v)3.2 was high in the INE, atrioventricular node and sino-atrial node, and that of Ca(v)3.2 high in Purkinje fibres. Although the expression of HCN1 and Ca(v)1.3 was low in the rat heart, it was relatively higher in the INE, atrioventricular node and sino-atrial node than in right atrial and right ventricular (working) myocytes. Both HCN2 and Ca(v)1.2 were expressed at higher levels in working myocytes than in nodal tissues and in the INE. Hierarchical clustering analysis demonstrated that the expression of the HCN and calcium channels in INE was similar to that in the slow-response automatic cells and different from that in working myocytes and Purkinje fibres. The expression of HCN and calcium channels in the INE of the adult rat heart is similar to that of slow-response automatic cells and provides a substrate for automatic phase 4 depolarization in cells.

[Electrophysiologic characteristics of Ito in myocytes from cardiac Koch triangle].

OBJECTIVE: To explore the electrophysiologic characteristics of Ito in myocytes from cardiac Koch triangle. METHODS: Patch clamp technique was employed to investigate the I-V and D-V relation, steady-state activation and inactivation kinetics of Ito from myocytes in Koch triangle of rabbit hearts. RESULTS: (1) The maximum peak current (pA) and peak current density (pA/pF) at +20 mV in PC (pacing cell), TC (transitonal cell) alpha,beta, AC (atrial cell) and PL (purkinje-like cell) cells were different from each other (P < 0. 05); The cells also had different current density (P < 0.05) except between TCalpha, TCbeta and PL cells (P > 0.05). (2) The half activation potential (V(mIto1/2), mV) of steady state activation among PC, TCalpha, TCbeta, AC and PL were significantly different (P < 0.05); The paired comparison between TCalpha and TCbeta, AC and PL, and TCbeta and PC showed significant difference (P < 0.05); but the differences between TCa and PC, TCbeta and AC, and TCbeta and PL were not significant (P > 0.05). (3) The half inactivation potential (V5(hIto1/2), mV) of steady state inactivation between TCalpha and PC, TCa and PL, TCbeta and PC, and TCbeta and PL demonstrated significant differences(P < 0.05). The differences of K(hIto) between TCalpha and TCbeta, PC and PL, TCbeta and PC, and AC and PL were significant (P < 0.05). CONCLUSIONS: Ito of cardiac cells from Koch triangle in rabbit hearts are heterogeneous, but relatively specified and distributed in different groups.

Sodium current kinetics of transitional myocytes in Koch triangle of rabbit hearts.

BACKGROUND: Few studies have explored the inward sodium current (INa) kinetics of transitional cardiomyocytes. This study aimed to explore the kinetics of transitional cardiomyocytes types alpha and beta. METHODS: The whole-cell patch clamp technique was used to study the rapid INa of isolated transitional cardiomyocytes in the Koch triangle of rabbit hearts. RESULTS: Maximal amplitude and density of INa in type alpha and type beta was (-1627 +/- 288) pA (alpha), (-35.17 +/- 6.56) pA/pF (beta) and (-3845 +/- 467) pA (alpha), (-65.64 +/- 10.23) pA/pF (beta) (P < 0.05). Steady state activation curves of INa, fitted to a Boltzmann distribution for both types, were sigmoid in shape. Half activation voltage and slope factors did not significantly differ between types at (-43.46 +/- 0.85) mV (alpha), (-41.39 +/- 0.47) mV (beta) or (9.04 +/- 0.66) mV (alpha), (11.08 +/- 0.89) mV (beta). Steady state inactivation curves of INa, fitted to a Boltzmann distribution in both types were inverse "S" shape. Half inactivation voltage and slope factors were (-109.9 +/- 0.62) mV (alpha), (-107.5 +/- 0.49) mV (beta) and (11.78 +/- 0.36) mV (alpha), (11.57 +/- 0.27) mV(beta), (P > 0.05), but time constants of inactivation were significantly different at (1.10 +/- 0.19) mV (alpha) and (2.37 +/- 0.33) ms (beta), (P < 0.05). Time constants of recovery from inactivation of INa for both types were (122.16 +/- 27.43) mV (alpha) and (103.84 +/- 28.97) ms (beta) (P < 0.05). CONCLUSIONS: Transitional cardiomyocytes in rabbit hearts show a heterogeneous, voltage gated and time dependent fast inward sodium current. Types alpha and beta show the features of INa similar to those in slow- and fast-response myocytes, with probably better automaticity and conductivity, respectively.

[Quantitative connexin mRNA detection in posterior nodal extension of adult rat heart].

OBJECTIVE: To quantitatively detect the expression of connexins (Cx) mRNA in the posterior nodal extension (PNE) of adult rat heart and understand the relationship between Cx expression and atrial ventricular nodal reentrant tachycardia (AVNRT). METHODS: PNE was separated from adult rat heart by means of laser microdissection (LCM), and the cells were also isolated from the atrioventricular node (AVN), sinoatrial node (SAN), Purkinje fiber (PF), right atrium (RA) and right ventricle (RV), to serve as the controls. The Cx mRNA level was detected in these cells with quantitative real-time PCR (QRT-PCR). RESULTS: The cells were successfully isolated from the PNE and other regions of adult rat heart, where heterogeneous expression of the 3 Cx isoforms (Cx43, Cx45, and Cx40) were observed. Cx45 mRNA showed higher expression in the PNE than in the working myocardium, whereas Cx43 mRNA level was about 25 times higher (P<0.05) in the working myocardium and 18 times higher (P<0.05) in the PF than in the PNE. In the PF, Cx40 mRNA level was proximately 6.8 times (P<0.01) as much as that in the PNE. Cx expression in the PNE was, however, similar to that in the SAN and AVN. CONCLUSION: Cx mRNAs exhibit heterogeneous expression in the PNE to allow the formation of the slow pathway. In addition, Cx expression in the PNE is very different from that in the adjacent myocardium, resulting in conduction discontinuity at the cellular junction, where, on certain occasion, unidirectional block may occur to cause AVNRT.

[An application of laser capture microdissection to isolate the pure atrial cells].

OBJECTIVE: To use the laser capture microdissection (LCM) to isolate the pure atrial cells from heart. To extract tiny amount RNA and amplify the genes of beta-actin and GAPDH. METHODS: First, the prepared sample RNA was extracted and verified. Second, the frozen sections were scraped after haematoxylin-eosin staining, and then the RNA of scraped tissues was extracted and verified. Last, the tiny amount RNA was extracted from atrial cells captured by LCM, and the genes of beta-actin and GAPDH were amplified by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The atrial cells isolated with LCM had clear morphology after quick staining. The high quality RNA was extracted from LCM sample and scraped tissues, of which A260/A280 was 1.9-2.0. And 18S rRNA and 28S rRNA were seen distinctly on the gel of RNA formaldehyde denaturing gel electrophoresis, which indicated that RNA didn't degrade before cells captured. Too small amount of RNA extracted from captured cells was below the detectable limit of gel electrophoresis or ultraviolet spectrophotometer. Bands of 300 bp and 357 bp could be amplified by RT-PCR from tiny amount RNA. CONCLUSION: Highly pure atrial cells could be obtained by LCM. Tiny amount RNA of captured atrial cells could be extracted and amplified with genes of beta-actin and GAPDH.

Morphological and electrophysiological properties of single myocardial cells from Koch triangle of rabbit heart.

BACKGROUND: The morphological and electrophysiological characteristics of cardiac cells in Koch triangle are still disputed. We studied the appearance and electrical properties of these diverse myocytes to elucidate their complex electrophysiological phenomena. METHODS: Experiments were conducted using cooled charge coupling device (CCD) system and whole cell, patch clamp technique to determine the morphology, action potential and sodium current density of single viable myocytes enzymatically isolated from the Koch triangle of rabbit hearts. RESULTS: Morphologically, cardiac cells in shape of spider, tiny spindle, slender spindle, rod and strip were observed in percentage of 3.0 +/- 0.3, 35.0 +/- 5.0, 15.0 +/- 2.0, 40.0 +/- 5.0 and 6.0 +/- 0.7 respectively. The cellular dimensions and capacitance gradually increased in the above order (all P < 0.05). Electrophysiologically, action potential configurations recorded from them were similar respectively to nodal (N), atrial nodal (AN), nodal Hisian (NH), atrial (A) and Hisian like potentials obtained from the intact atrioventricular nodal preparations. Diastolic depolarization appeared in all myocytes except for rod cells. Sodium current density increased in the order of tiny spindle, strip, rod, slender spindle cell (all P < 0.05), but could not be detected in spider-shaped cells. Linear regression analysis revealed that membrane capacitance was correlated negatively to the rate of diastolic depolarization r = -0.70, P < 0.001, but positively to maximum depolarization potential, amplitude of action potential, upstroke velocity and maximum peak value of sodium current density r = 0.84, 0.80, 0.87 and 0.75, respectively; all P < 0.001. CONCLUSIONS: The results demonstrated that spider-shaped, spindle, rod and strip cells in Koch triangle might correspond to pacemaking, transitional, atrial and Purkinje like cells, respectively. Furthermore, tiny spindle and slender spindle cells were referred to transitional cell alpha (TCalpha) and beta (TCbeta) accordingly considering their distinctive electrical properties. Different myocytes with diverse electrical properties constituted the infrastructure of sophisticated electrophysiological phenomena in Koch triangle. In view of the prominent percentage and electrical properties, tiny spindle and slender spindle cells were presumed to play important roles.

[Expression of L-type calcium channel alpha1C subunit in adult rat heart].

OBJECTIVE: To investigate the expression and distribution of L-type calcium channel alpha1C subunits in adult rat heart. METHODS: HE staining was applied on the frozen sections of adult rat heart to identify the sinoatrial node (SAN), atrioventricular node (AVN), and posterior nodal extension (PNE). The protein expression of L-type calcium channel alpha1C in adult rat heart and its cellular localization were examined by Western blotting and immunohistochemistry, respectively. RESULTS: L-type calcium channel alpha1C subunit was immunolocalized on the membrane of the myocardial cells, and its expression increased gradually in the SAN, AVN, PNE, right atrium and right ventricle. The protein level of L-type calcium channel alpha1C in the AVN was similar to that in the PNE (P>0.05), and its level in the right atrium and ventricle were significantly higher than those in the SAN and AVN (P<0.01). The results of Western blotting were well consistent with the results of immunohistochemistry. CONCLUSION: The expression of L-type calcium channel alpha1C subunit may play a role in the electrophysiological functions of the heart.