Search

Objective: This study determined whether the GENECUBE rapid nucleic acid amplification test could directly detect nuc and mecA genes in clinical blood culture samples of Staphylococcus and various other pathogens., Methods: Between September 2020 and December 2021, 537 blood culture samples from 192 patients with suspected bacteremia were tested using conventional assays (MicroScan WalkAway96 or VITEK 2 systems) and GENECUBE nuc and mecA assays. Isolates from samples with discrepant results between the conventional and GENECUBE assays were further evaluated using MALDI-TOF mass spectrometry, disk diffusion testing using cefoxitin, broth microdilution testing using oxacillin, and sequencing for mecA. Bacterial solutions containing a mixture of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) were prepared to evaluate the limit of detection (LOD) of mecA., Results: Using conventional assays as the reference, the sensitivity, specificity, and positive and negative predictive values (95 % confidence interval) of GENECUBE were 100 % (96.8-100 %), 100 % (99.1-100 %), 100 % (96.8-100 %), and 100 % (99.1-100 %), respectively, for nuc detection and 100 % (96.1-100 %), 98.9 % (97.4-99.6 %), 94.9 % (88.5-98.3 %), and 100 % (99.2-100 %), respectively, for mecA detection. Sequencing analysis of five samples identified as methicillin-sensitive staphylococci using conventional assays and methicillin-resistant staphylococci using GENECUBE revealed the presence of methicillin-resistant isolates in all samples. The estimated LOD of mecA was 10 4 colony-forming units (CFU)/mL of MRSE with GENECUBE, compared with 10 5  CFU/mL with conventional assays., Conclusion: The GENECUBE assay accurately detected mecA in positive blood culture samples and had higher sensitivity than conventional assays., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yoshihiro Soya is an employee of TOYOBO Co., Ltd., a private company that manufactures the GENECUBE analyzer and related reagents., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Xenopus young tadpoles regenerate a limb with the anteroposterior (AP) pattern, but metamorphosed froglets regenerate a hypomorphic limb after amputation. The key gene for AP patterning, shh, is expressed in a regenerating limb of the tadpole but not in that of the froglet. Genomic DNA in the shh limb-specific enhancer, MFCS1 (ZRS), is hypermethylated in froglets but hypomethylated in tadpoles: shh expression may be controlled by epigenetic regulation of MFCS1. Is MFCS1 specifically activated for regenerating the AP-patterned limb? We generated transgenic Xenopus laevis lines that visualize the MFCS1 enhancer activity with a GFP reporter. The transgenic tadpoles showed GFP expression in hoxd13-and shh-expressing domains of developing and regenerating limbs, whereas the froglets showed no GFP expression in the regenerating limbs despite having hoxd13 expression. Genome sequence analysis and co-transfection assays using cultured cells revealed that Hoxd13 can activate Xenopus MFCS1. These results suggest that MFCS1 activation correlates with regeneration of AP-patterned limbs and that re-activation of epigenetically inactivated MFCS1 would be crucial to confer the ability to non-regenerative animals for regenerating a properly patterned limb., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Agonists for TLR9 and stimulator of IFN genes (STING) offer therapeutic applications as both anti-tumor agents and vaccine adjuvants, though their clinical applications are limited; the clinically available TLR9 agonist is a weak IFN inducer and STING agonists induce undesired type 2 immunity. Yet, combining TLR9 and STING agonists overcame these limitations by synergistically inducing innate and adaptive IFNγ to become an advantageous type 1 adjuvant, suppressing type 2 immunity, in addition to exerting robust anti-tumor activities when used as a monotherapeutic agent for cancer immunotherapy. Here, we sought to decipher the immunological mechanisms behind the synergism mediated by TLR9 and STING agonists and found that their potent anti-tumor immunity in a Pan02 peritoneal dissemination model of pancreatic cancer was achieved only when agonists for TLR9 and STING were administered locally, and was via mechanisms involving CD4 and CD8 T cells as well as the co-operative action of IL-12 and type I IFNs. Rechallenge studies of long-term cancer survivors suggested that the elicitation of Pan02-specific memory responses provides protection against the secondary tumor challenge. Mechanistically, we found that TLR9 and STING agonists synergistically induce IL-12 and type I IFN production in murine APCs. The synergistic effect of the TLR9 and STING agonists on IL-12p40 was at protein, mRNA and promoter activation levels, and transcriptional regulation was mediated by a 200 bp region situated 983 bp upstream of the IL-12p40 transcription initiation site. Such intracellular transcriptional synergy may hold a key in successful cancer immunotherapy and provide further insights into dual agonism of innate immune sensors during host homeostasis and diseases., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Genetically encoded calcium indicators (GECIs) are widely used to measure calcium transients in neuronal somata and processes, and their use enables the determination of action potential temporal series in a large population of neurons. Here, we generate a transgenic mouse line expressing a highly sensitive green GECI, G-CaMP9a, in a Flp-dependent manner in excitatory and inhibitory neuronal subpopulations downstream of a strong CAG promoter. Combining this reporter mouse with viral or mouse genetic Flp delivery methods produces a robust and stable G-CaMP9a expression in defined neuronal populations without detectable detrimental effects. In vivo two-photon imaging reveals spontaneous and sensory-evoked calcium transients in excitatory and inhibitory ensembles with cellular resolution. Our results show that this reporter line allows long-term, cell-type-specific investigation of neuronal activity with enhanced resolution in defined populations and facilitates dissecting complex dynamics of neural networks in vivo., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Abstract: Ozonated water is a possible hand washing alternative to antimicrobial soap and water. In a previous report, 4 ppm of ozonated water removed artificially contaminated bacteria from the hands of healthy volunteers as effectively as antimicrobial or nonantimicrobial soap and water. Currently, there is a lack of data on the efficacy of ozonated water in removing bacteria from hands loaded with organic materials. This study aimed to evaluate the effectiveness of ozonated water in removing bacteria from hands contaminated with organic material, according to the American Society for Testing and Materials E2946-13. Sixteen healthy volunteers were randomly assigned to the ozonated water group and the antimicrobial soap and water group. Their hands were contaminated with an avirulent strain of Escherichia coli in beef broth suspension. Approximately 3-log CFU bacterial reductions between baseline and postwash colonies were observed on the hands in both groups. Ozonated water may remove bacteria from hands contaminated with organic material with similar effectiveness as antimicrobial soap and water., (Copyright ©, International Association for Food Protection.)

Vaccine adjuvants are traditionally used to augment and modulate the immunogenicity of vaccines, although in many cases it is unclear which specific molecules contribute to their stimulatory activity. We previously reported that both subcutaneous and intranasal administration of hydroxypropyl-β-cyclodextrin (HP-β-CD), a pharmaceutical excipient widely used to improve solubility, can act as an effective adjuvant for an influenza vaccine. However, the mechanisms by which mucosal immune pathway is critical for the intranasal adjuvant activity of HP-β-CD have not been fully delineated. Here, we show that intranasally administered HP-β-CD elicits a temporary release of IL-33 from alveolar epithelial type 2 cells in the lung; notably, IL-33 expression in these cells is not stimulated following the use of other vaccine adjuvants. The experiments using gene deficient mice suggested that IL-33/ST2 signaling is solely responsible for the adjuvant effect of HP-β-CD when it is administered intranasally. In contrast, the subcutaneous injection of HP-β-CD and the intranasal administration of alum, as a damage-associated molecular patterns (DAMPs)-inducing adjuvant, or cholera toxin, as a mucosal adjuvant, enhanced humoral immunity in an IL-33-independent manner, suggesting that the IL-33/ST2 pathway is unique to the adjuvanticity of intranasally administered HP-β-CD. Furthermore, the release of IL-33 was involved in the protective immunity against influenza virus infection which is induced by the intranasal administration of HP-β-CD-adjuvanted influenza split vaccine. In conclusion, our results suggest that an understanding of administration route- and tissue-specific immune responses is crucial for the design of unique vaccine adjuvants., (Copyright © 2020 Kobari, Kusakabe, Momota, Shibahara, Hayashi, Ozasa, Morita, Matsumoto, Saito, Ito, Kuroda and Ishii.)

Extracellular host-derived DNA, as one of damage associated molecular patterns (DAMPs), is associated with allergic type 2 immune responses. Immune recognition of such DNA generates the second messenger cyclic GMP-AMP (cGAMP) and induces type-2 immune responses; however, its role in allergic diseases, such as asthma, has not been fully elucidated. This study aimed to determine whether cGAMP could induce asthma when used as an adjuvant. We intranasally sensitized mice with cGAMP together with house dust mite antigen (HDM), followed by airway challenge with HDM. We then assessed the levels of eosinophils in the broncho-alveolar lavage fluid (BALF) and serum HDM-specific antibodies. cGAMP promoted HDM specific allergic asthma, characterized by significantly increased HDM specific IgG1 and total IgE in the serum and infiltration of eosinophils in the BALF. cGAMP stimulated lung fibroblast cells to produce IL-33 in vitro , and mice deficient for IL-33 or IL-33 receptor (ST2) failed to develop asthma enhancement by cGAMP. Not only Il-33 -/- mice, but also Sting -/- , Tbk1 -/- , and Irf3 -/- Irf7 -/- mice which lack the cGAMP-mediated innate immune activation failed to increase eosinophils in the BALF than that from wild type mice. Consistently, intranasal and oral administration of amlexanox, a TBK1 inhibitor, decreased cGAMP-induced lung allergic inflammation. Thus, cGAMP functions as a type 2 adjuvant in the lung and can promote allergic asthma in manners that dependent on the intracellular STING/TBK1/IRF3/7 signaling pathway and the resultant intercellular signaling pathway via IL-33 and ST2 might be a novel therapeutic target for allergic asthma., (Copyright © 2019 Ozasa, Temizoz, Kusakabe, Kobari, Momota, Coban, Ito, Kobiyama, Kuroda and Ishii.)

Leukotriene B 4 receptor 1 (BLT1) triggers the migration of granulocytes and activated T cells; however, its role in B-cell function remains unclear. Here we report that BLT1 is required to induce the production of antigen-specific IgA against oral vaccine through mediating innate immune signals from commensal bacteria. B cells acquire BLT1 expression during their differentiation to IgA + B cells and plasma cells in Peyer's patches and the small intestinal lamina propria, respectively. BLT1 KO mice exhibited impaired production of antigen-specific fecal IgA to oral vaccine despite normal IgG responses to systemically immunized antigen. Expression of MyD88 was decreased in BLT1 KO gut B cells and consequently led to diminished proliferation of commensal bacteria-dependent plasma cells. These results indicate that BLT1 enhances the proliferation of commensal bacteria-dependent IgA + plasma cells through the induction of MyD88 and thereby plays a key role in the production of antigen-specific intestinal IgA.

Recently, it was reported that 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD), a common pharmaceutical additive, can act as a vaccine adjuvant to enhance protective type-2 immunogenicity to co-administered seasonal influenza split vaccine by inducing host-derived damage-associated molecular patterns (DAMPs). However, like most other DAMP-inducing adjuvants such as aluminum hydroxide (Alum), HP-β-CyD may not be sufficient for the induction of protective type-1 (cellular) immune responses, thereby leaving room for improvement. Here, we demonstrate that a combination of HP-β-CyD with a humanized TLR9 agonist, K3 CpG-ODN, a potent pathogen-associated molecular pattern (PAMP), enhanced the protective efficacy of the co-administered influenza split vaccine by inducing antigen-specific type-2 and type-1 immune responses, respectively. Moreover, substantial antigen-specific IgE induction by HP-β-CyD, which can cause an allergic response to immunized antigen was completely suppressed by the addition of K3 CpG-ODN. Furthermore, HP-β-CyD- and K3 CpG-ODN-adjuvanted influenza split vaccination protected the mice against lethal challenge with high doses of heterologous influenza virus, which could not be protected against by single adjuvant vaccines. Further experiments using gene deficient mice revealed the unique immunological mechanism of action in vivo , where type-2 and type-1 immune responses enhanced by the combined adjuvants were dependent on TBK1 and TLR9, respectively, indicating their parallel signaling pathways. Finally, the analysis of immune responses in the draining lymph node suggested that HP-β-CyD promotes the uptake of K3 CpG-ODN by plasmacytoid dendritic cells and B cells, which may contributes to the activation of these cells and enhanced production of IgG2c. Taken together, the results above may offer potential clinical applications for the combination of DAMP-inducing adjuvant and PAMP adjuvant to improve vaccine immunogenicity and efficacy by enhancing both type-2 and type-1 immune responses in a parallel manner.

Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nasal vaccination has the potential to elicit systemic and mucosal immunity against pathogens. However, split and subunit vaccines lack potency at stimulating mucosal immunity, and an adjuvant is indispensable for eliciting potent mucosal immune response to nasal vaccines. Endocine, a lipid-based mucosal adjuvant, potentiates both systemic and mucosal immune responses. Although Endocine has shown efficacy and tolerability in animal and clinical studies, its mechanism of action remains unknown. It has been reported recently that endogenous danger signals are essential for the effects of some adjuvants such as alum or MF59. However, the contribution of danger signals to the adjuvanticity of Endocine has not been explored. Here, we show that RNA is likely to be an important mediator for the adjuvanticity of Endocine. Administration of Endocine generated nucleic acids release, and activated dendritic cells (DCs) in draining lymph nodes in vivo. These results suggest the possibility that Endocine indirectly activates DCs via damage-associated molecular patterns. Moreover, the adjuvanticity of Endocine disappeared in mice lacking TANK-binding kinase 1 (Tbk1), which is a downstream molecule of nucleic acid sensing signal pathway. Furthermore, co-administration of RNase A reduced the adjuvanticity of Endocine. These data suggest that RNA is important for the adjuvanticity of Endocine.

Intranasal vaccination with inactivated influenza viral antigens is an attractive and valid alternative to currently available influenza (flu) vaccines; many of which seem to need efficient and safe adjuvant, however. In this study, we examined whether hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used pharmaceutical excipient to improve solubility and drug delivery, can act as a mucosal adjuvant for intranasal flu vaccines. We found that intranasal immunization of mice with hemagglutinin split- as well as inactivated whole-virion influenza vaccine with HP-β-CD resulted in secretion of antigen-specific IgA and IgGs in the airway mucosa and the serum as well. As a result, both HP-β-CD adjuvanted-flu intranasal vaccine protected mice against lethal challenge with influenza virus, equivalent to those induced by experimental cholera toxin-adjuvanted ones. Of note, intranasal use of HP-β-CD as an adjuvant induced significantly lower antigen-specific IgE responses than that induced by aluminum salt adjuvant. These results suggest that HP-β-CD may be a potent mucosal adjuvant for seasonal and pandemic influenza vaccine., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A 53-year-old man was diagnosed as having idiopathic thrombocytopenic purpura (ITP). Hematochezia appeared under steroid therapy for ITP after the diagnosis of ITP 18 months. Colonoscopic study demonstrated inflamed rectal mucosa but there was no evidence of infectious colitis. The colonoscopic and pathological findings were compatible with ulcerative colitis (UC). There have been a few reports of patients with UC complicated by ITP, but in all except one report, UC preceded ITP. This is the third case in which ITP preceded UC and the first case in which the proctitis type of UC was complicated by ITP in Japan.

A 21-year-old male patient with non-A, non-B, non-C acute hepatitis was complicated by hepatitis-associated severe aplastic anemia during hospitalization for active hepatitis. He was promptly diagnosed and treated with methylprednisolone, anabolic steroids, cyclosporin A, granulocyte colony-stimulating factor (G-CSF), and antithymocyte globulin (ATG). He responded quickly to the immuno-suppressive therapy and was transfusion independent after 25 days and granulocyte colony-stimulating factor independent at 57 days after ATG therapy. Although the etiology of hepatitis-associated aplastic anemia is still controversial, the authors emphasized the importance to carefully follow non-A, non-B, non-C hepatitis patients without aplastic anemia for more than three months after hepatitis episodes in order to improve outcome of this lethal disease.

Epstein-Barr virus associated T-Cell lymphoma mimicking malignant histiocytosis (MH) has been previously reported. We selected 10 autopsy cases of extranodal lymphoma or histiocytic tumor, which showed an EBV presence in the tumor cells as well as a fulminant clinical course. The detailed clinicopathologic features were thus clarified. A retrospective study was performed on ten adult patients, eight males and two females, and almost all cases presented with a fulminant clinical course, revealing pancytopenia, liver dysfunction and disseminated intravascular coagulopathy. Immunophenotypic and genotypic studies along with in situ hybridization (ISH) were performed. The autopsy findings mainly showed extra nodal involvement in the liver (10 patients), spleen (9 patients), intestinal tract (5 patients), bone marrow (5 patients), nasal cavity, lungs, adrenal glands, kidneys (2 patients) and brain. Histologically atypical pleomorphic lymphoid cells were observed to infiltrate with reactive histiocytes, some of which showed hemophagocytosis. Based on the histological and clinical findings, diagnosis of malignant histiocytosis was made. ISH showed an EBV-presence in almost all the tumor cells. The immunophenotype and/or genotype studies demonstrated T-cell lymphoma (2 patients), Histiocytic tumors (2 patients), B-cell lymphoma (1 patients), natural killer (NK) cell lymphoma (3 patients), and T/NK lymphoma (2 patients), in which T or NK could not be confirmed, due to a lack of fresh materials. Based on the above findings, the histological appearance of EBV-associated MH previously defined was shown to be common to extra-nodal malignant lymphomas having origin in various organs, although the cytological and genetic features were heterogenous.

Malignant histiocytosis has been described as a proliferation of morphologically atypical histiocytes, but it is difficult to determine whether or not malignant proliferation is present based on morphology alone. Recently the disorder has been thought to be heterogeneous, and therefore a true histiocytic origin is considered to be rare. The Epstein-Barr virus (EBV) is thought to have the ability to transform human cells. Therefore, eight cases of malignant histiocytic (MH) syndrome and five cases of virus-associated hemophagocytic syndrome (VAHS) were analyzed using a polymerase chain reaction (PCR) and the in situ hybridization (ISH) method in order to determine their relationship to EBV infection. At the same time, the cellular origin of these syndromes was also studied. The results indicated that three of the MH cases were derived from T cells while four the MH cases were from histiocytes. The amplification of the EBV-LYDMA region, which was used to determine the monoclonality, was detected in two MH cases and one VAHS case, and all these cases showed only one band. An ISH study also demonstrated the presence of an EBV in these three cases. One of the EBV-positive cases revealed an amplification of the EBV-LYDMA region by the PCR method before showing any sign of MH clinically. In the VAHS cases, the EBV genome was detected in hemophagocytic cells. The EBV-positive cases all demonstrated a rapid clinical course. Based on these results it is possible that EBV infection causes similar rapid clinical features in some cases of both MH and VAHS by the same mechanism.

Epstein-Barr virus (EBV) has a particular propensity for B lymphocytes, but in a few cases it seems to play a role in histiocytic disorders and EVB DNA has been identified in histiocytes. To determine what kind of cell proliferate clonally, we studied a patient with malignant histiocytosis that developed after chronic EBV infection. Polymerase chain reaction (PCR) for lymphocyte-defined membrane antigen (LYDMA) of EBV, a marker of monoclonality, double stainings of cell markers (B, T lymphocytes; histiocytes), and in situ hybridization for EBV were performed in tissues obtained in 1987 and 1990 before the appearance of malignant histiocytosis and in 1991 after the disease was diagnosed. PCR for LYDMA from multiple samples during the disease showed the same single band, indicating that chronic EBV infection and malignant histiocytosis were caused by the same single virion. We also found a single terminal repeat band of EBV which supports this finding. In the studies of double stainings, EBV was present in histiocytes of the non-neoplastic early stage, and in the neoplastic cells of malignant histiocytosis. The histiocyte, infected with EBV, clonally expanded to result in malignant histiocytosis.

We used the polymerase chain reaction (PCR) in situ hybridization (ISH) (PCR-ISH) on sections of malignant lymphoma and nonspecific lymphadenitis to detect small amounts of Epstein-Barr virus (EBV), a DNA virus of the herpes virus family. We first surveyed the EBV DNA by Southern blot analysis and PCR, and then compared results of the two PCR/ISH methodologies with the results of simplified/sensitive ISH for the positive cases. The target of the simplified in situ (DNA-ISH) was a few copies of EBV DNA per cell, and the target of the sensitive in situ (RNA-ISH) was as many as 10(7) copies of EBV RNA per cell. When EBV DNA was detected by Southern blot, DNA-ISH, RNA-ISH and PCR-ISH all revealed EBV genomes. When PCR revealed only amplified EBV DNA, DNA-ISH showed no EBV genomes, but PCR-ISH and RNA-ISH showed EBV genomes in a few cells. When PCR showed no detectable amplified EBV DNA, all of DNA-ISH, RNA-ISH and PCR-ISH showed no genomes. These findings indicate that PCR-ISH consistently detected a few copies of the EBV virions. The PCR-ISH was as sensitive as RNA-ISH. The RNA-ISH could not detect virus if RNA was not expressed, but the PCR-ISH could detect virus without such expression. The ability to detect a single copy of a specific gene in situ has many advantages and multiple applications in molecular biology, pathology, and cell biology.

To better characterize the clonality and pathogenesis of Hodgkin's disease (HD), we used polymerase chain reaction (PCR) and Southern blot to analyze the rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes, the bcl-2 oncogene, and the Epstein-Barr virus (EBV) genotype. In situ hybridization studies of EBV were also done. Twenty-six cases of HD were compared with 15 cases of non-specific lymphadenitis, 7 with incipient adult T-cell leukemia/lymphoma (ATLL), and 4 T-cell rich B-cell lymphomas (TRBL), all of which histologically resembled HD. EBV genes were detected in 20 of 26 HD patients (77%) and in 7 of 15 patients with non-specific lymphadenitis (47%), 5 of 7 with incipient ATLL (71%), and 1 of 4 with TRBL (25%). In contrast to specimens of non-specific lymphadenitis, TRBL, and incipient ATLL, only one EBV genotype was evident in the specimens of HD. EBV latent membrane protein (LMP) was detected immunologically in 16 of 26 HD patients (62%), one of four TRBL (25%) and one of seven incipient ATLL (14%), but it was not evident in non-specific lymphadenitis. The LMP positive cases showed amplified EBV genomes. Only one of the 26 cases of HD had a bcl-2 gene rearrangement by PCR, but this was not seen in any other disease. The bcl-2 protein was detected immunologically in seven of the 26 HD patients (27%) and in one of the seven incipient ATLL cases (14%). EBV has been reported to upregulate bcl-2 expression, but in this study the presence of bcl-2 protein did not correlate with the presence of the t(14;18) translocation or EBV-LMP. All TRBLs showed rearrangement of the immunoglobulin genes by PCR and/or Southern blot, and the giant cells were of B-cell type. All incipient ATLLs displayed rearrangement of the TCR genes, and the giant cells were of T-cell origin. In seven of 26 HD cases, the giant cells were weakly stained with T-cell antibodies, in another seven positive with B-cell antibodies and in 18 instances polyclonally positive for both kappa and lambda. However, PCR and Southern blot displayed only two cases of TCR gene rearrangement, while two others had very weak rearrangements of immunoglobulin gene positive only by PCR. Thus the T and B-cell genotype did not correlate with the T and B-cell phenotype recorded in these cases. The absence of Ig and TCR gene rearrangements seems to be common in HD, compared with in TRBL and incipient ATLL.

In order to thoroughly characterize the clonal population of lymphoid hyperplasia of the orbit and conjunctiva, we investigated six cases which were histologically proven to be benign lymphoid hyperplasia. We analyzed the clonal rearrangements of the antigen receptors and bcl-2 gene, Epstein-Barr virus (EBV), and human T-cell leukemia virus type 1 (HTLV-I) by Southern blot and/or polymerase chain reaction (PCR), and performed in situ hybridization for mRNA of kappa and lambda immunoglobulin. Five cases showed rearrangements of immunoglobulin heavy chain gene (JH) and/or light chain gene (J kappa), and the monoclonal V-J recombination of JH in PCR. However, the rearranged bands were much more faint than was the germ-line band. We considered the monoclonal population of B cells small. Two of the five cases recurred locally after four and nine years respectively. Because benign lymphoid hyperplasias frequently contain an occult monoclonal B-cell population, a follow-up should be conducted. The remaining case in our investigation showed a rearrangement of the T-cell-receptor gene and proviral DNA of HTLV-I, and it showed rapid progress to adult T-cell leukemia after the biopsy. EBV and bcl-2 gene rearrangements were not observed in any of the six cases we studied.

The nasopharyngeal lymphoepithelioma is closely related with Epstein-Barr virus. The first clinical manifestation is frequently enlargement of the cervical lymph nodes of unknown origin. The histology of the metastatic lymphoepithelioma in the lymph nodes sometimes resembles those of other metastatic carcinoma and granulomatous diseases. To differentiate the lymphoepithelioma from other disorders, we detected Epstein-Barr virus (EBV), using stamp specimens. By polymerase chain reaction, all seven lymphoepitheliomas presented amplified EBV genomes, while five of the 58 other carcinomas and eight of the 19 granulomatous diseases also did. By in situ hybridization, lymphoepitheliomas showed EBV genomes which were confined to the tumor cells, though in the other diseases they were found in the lymphocytes. Detection of EBV is very useful in making a diagnosis of lymphoepitheliomas.

Immunophenotyping of lymphoma using paraffin-embedded lymphoid tissue is useful in identifying the large neoplastic B cells in T-cell-rich B-cell lymphoma (TRBL), but does not succeed in deciding clonality. We studied six cases to determine the clonal population of B and T cells of TRBL. Immunohistochemistry on frozen and paraffin-embedded material showed that the cellular population in all six cases consisted mainly of T cells; fewer than ten percent of the cells stained as B cells. However, in all cases, monoclonality of the immunoglobulin was helpful for diagnosing the B-cell neoplasia. Southern blot-yielded genetic analysis showed monoclonality of B cells in three cases, but no evidence of clonality in the T cells. Moreover, gene monoclonality has been detected in all cases examined by polymerase chain reaction, using the primers for the V and J regions of the immunoglobulin heavy chain gene. For T cells, the D and J regions of the T-cell receptor (TCR) beta chain showed the same patterns of oligoclonal bands in all cells, and the V and J regions of the TCR gamma chain showed the same bands in all. The expression of TCR V beta families was polyclonal but restricted.

The cervical lymph nodes of 27 patients with histiocytic necrotizing lymphadenitis (HNL) were examined, as were those of 9 patients with tuberculous lymphadenitis (Tb), 10 with reactive paracortical hyperplasia (RPH), and 10 with nonspecific lymphadenitis (NSL). Southern blot analysis, the polymerase chain reaction (PCR), and in situ hybridization were use to locate the human herpesvirus-6 (HHV-6) genome. Southern blot analysis showed that all cases were negative for HHV-6 genomes, although all but one HNL case expressed HHV-6 genome using PCR. On in situ hybridization all 10 HNL cases, 6 of the 10 RPH cases, 6 of the 10 NSL cases, and 2 of the 9 Tb cases showed HHV-6 DNA. These results indicate that the presence of HHV-6 genome is not specifically related to HNL, and that this virus could hibernate in a latent form in the cervical lymph nodes. In addition, we examined three different primers (A, B, and C) for PCR amplification of HHV-6 genomes.

Adult T cell leukemia/lymphoma (ATLL) is a mature T cell malignancy, especially derived from the CD4 positive T cell. To characterize the T cell, we examined the representation of T cell antigen receptor variable region, using the monoclonal antibodies [beta V 5 (a), beta V 5 (b), beta V 6 (a), beta V 8 (a), beta V 12 (a), alpha V 2 (a), alpha-beta V (a)]. Clinicopathologically we classified the lymph nodes of patients with ATLL into three states (1) human T cell leukemia virus type I (HTLV-I) associated lymphadenitis, reactive state; (2) incipient ATLL, early or pre-neoplastic state; and (3) ATLL, neoplastic state. The lymph nodes of all three states were composed of unvarying CD4 positive T cells. Most of the lymph nodes with ATLL consistently presented alpha V 2 antigen, but no others. In HTLV-I associated lymphadenitis, only a few cells reacted for alpha V 2, as in non-specific lymphadenitis without ATLL features. One of three cases with incipient ATLL presented alpha V 2. The selected expression of T cell antigen receptor V region might be associated with the presence of HTLV-I encoded superantigen, similar to human immunodeficiency virus (HIV).

Using the polymerase chain reaction (PCR) to examine the occurrence of bcl-2/JH joining produced by t(14;18) chromosomal translocation, amplified DNA was detected in 2 of 18 lymph nodes showing reactive lymphadenopathy. The PCR was repeated in these two lymph nodes using the same DNA samples, but no amplification was detected at the second attempt. Thus the amplified DNA was considered to be derived from one copy of joined bcl-2/JH in one cell, or from a few copies in a few clonal cells with the same joined bcl-2/JH. These results suggest that false joining of bcl-2/JH at the t(14;18) junction may occur in reactive lymph nodes.

Five patients with histiocytic necrotizing lymphadenitis (Kikuchi's disease) with erythematous or popular skin lesions were studied. All patients healed naturally within a few months like the patients with no skin involvement. Histological findings for the skin lesions mimicked cutaneous malignant lymphoma. The infiltrated mononuclear cells usually demonstrated positive reactions for Ki-M1p (20-63%), lysozymes (13-54%), MT-1 (18-64%), UCHL-1 (22-39%) and LN2 (17-36%). OPD-4 and L26 positive cells were few in number. These results suggest that the infiltrated cells in a Kikuchi's disease skin lesion are composed of the same components as the affected lesion in the lymph node.

Histopathologic diagnosis of the bone marrow in leukemia is usually a supplementary method to the cytological in acute and chronic leukemia. However, for patients with MDS and MPD and with dry tap bone marrow biopsy is very important. Important morphological findings and useful immunohistochemical methods for differentiation and characterization of leukemia are reported and the usefulness of sequential examination of bone marrow in leukemia during and after chemotherapy is emphasized. In addition to leukemia, histological features and differential points of myelodysplastic syndrome (MDS) and myeloproliferative disorders (MPD) are mentioned. The proliferating megakaryocytes differed in size and shape between MDS and MPD. The difference in proliferating rate of the cells examined by PCNA was also useful to differentiate the two disorders histologically.

Human T-cell leukemia virus type I (HTLV-I) is associated with adult T-cell leukemia/lymphoma (ATLL). To examine the relationship between defective HTLV-I proviruses and clinicopathological features, we examined 95 patients with ATLL showing clonal integration of HTLV-I proviral DNA; 77 patients (81%) showed 1 clonal band, 15 (16%) showed 2 clonal bands, and 3 (3%) showed 3 clonal bands. In addition, the defective proviral form was detected in 28 patients (29%): 23 (30%) of the 77 with 1 clonal band, 4(27%) of the 15 with 2 clonal bands, and 1(33%) of the 3 with 3 clonal bands. The numbers of clonal bands had no association with the presence of defective proviruses. We classified the 95 patients with ATLL into four types according to clinicopathological features (smoldering leukemia, chronic leukemia, acute leukemia, and lymphoma types). The distribution of patients with the defective form was not different among these four types. The HTLV-I genomes must have integrated into the human genome DNA and been deleted partially in the cells. The defective form was kept during the clinical stage. All patients with the defective form showed defect of the gag or/and env region. No patient had a defect of the pX region. These data suggest that the pX region of HTLV-I must have played an important role in ATLL genesis.

Both the polymerase chain reaction (PCR) and Southern blot analysis were used to detect bcl-2 gene rearrangement in B-cell lymphoma. Recent molecular studies have shown that the translocation of karyotypic abnormalities t(14;18) results in the juxtaposition of the candidate proto-oncogene bcl-2 on chromosome 18 with the immunoglobulin heavy-chain locus on chromosome 14. We detected the bcl-2 rearrangement in three of six follicular lymphomas (50%), two of five follicular and diffuse lymphomas (40%), one of 13 diffuse medium-sized cell lymphomas (7.7%) and two of 33 diffuse large cell lymphomas (6.0%) through Southern blot analysis. With PCR, the rearrangement was demonstrated in five of eight follicular (63%), three of five follicular and diffuse (60%), seven of 36 diffuse large cell lymphomas (19%) and two of 13 diffuse medium-sized lymphomas (15%). Diffuse large cell lymphomas with bcl-2 rearrangement detected by PCR have a good prognosis as do cases of follicular lymphoma The bcl-2 gene is closely related to follicular lymphoma as described in previous reports and has general prognostic importance in diffuse large cell lymphoma of B cell type.

The clinical and histological findings of a 10-year-old girl with virus-associated haemophagocytic syndrome are presented. The serum levels of Epstein-Barr viral antigens were elevated. Epstein-Barr virus (EBV) genome was detected by polymerase chain reaction in bone marrow and lymph node specimens. Histologically, haemophagocytic histiocytes were present in bone marrow, and areas of non-suppurative necrosis were present in lymph nodes, where silver grain deposition of the EBV genome was demonstrated by in situ hybridization.