Monoclonal antibodies to a CD4 peptide derivative which includes the region corresponding to an immunoglobulin CDR3: evidence of the involvement of pre-CDR3-related region in HIV-1 and host cell interaction.

A CD4 peptide of amino acid residues 68-130 [CD4(68-130)], which had the capacities to inhibit HIV-1 replication and HIV-1-induced syncytium formation, was used as an immunogen for the preparation of mAb. The mAbs prepared were classified into at least five types (I-V) in terms of their recognition sites by ELISA using various kinds of smaller CD4 peptides. Among them, the type I mAb no. 35 recognizing amino acid residues 72-84, which lies just before the region corresponding to an immunoglobulin third complementarity-determining region (CDR3), showed the strongest effects in reducing both HIV-1 infection and HIV-1-induced syncytium formation, although a large amount of no. 35 mAb was necessary to reduce such HIV-1 activities compared with those of anti-Leu-3a and OKT4A mAbs which recognize CD4 epitopes near a portion corresponding to an immunoglobulin CDR2. Western blot analysis showed that the reactivities of CD4 molecule in CD4-positive cells or sCD4 molecule with types I-V mAbs were stronger than that with anti-Leu-3a mAb. Flow cytometry showed that no. 35 mAb was faintly reactive with native CD4 molecule on cell surface at the concn showing the inhibitory effects on HIV-1 infection and syncytium formation. In addition, a smaller peptide CD4(66-92), one of the good epitope peptides for no. 35 mAb, also showed strong inhibitory effect on HIV-1 infection as well as a weaker inhibitory effect on syncytium formation. These results suggest that, in addition to the CD4 CDR2-related region, the pre-CDR3-related region is also involved in the early events of the interactions between the host cell and HIV-1.     

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

 The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (K_ATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K _i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATP_i) equipotently in the presence and in the absence of intracellular Mg²⁺ (Mg²⁺ _i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATP_i only in the presence of Mg²⁺ _i. Uridine 5’-diphosphate (UDP) antagonized the ATP_i-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATP_i-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac K_ATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the K_ATP channel. Received: 24 October 1997 / Received after revision and accepted: 4 December 1997     

C to T transition at the first nucleotide of codon 63 of the β-globin gene corresponding to hemoglobin M-Saskatoon in an Indonesian boy

Summary Hemoglobin (Hb) M-Saskatoon, a variant of methemoglobin, is characterized by mild hemolysis. It is caused by the substitution of a histidine by a tyrosine at the 63rd amino acid residue of the -globin chain. Amplification and sequence analysis of genomic -globin DNA from an Indonesian boy diagnosed as having the more severe disease thalasemia demonstrated the presence of a C to T transition at nucleotide 473 in one of the two -blogin genes resulting in a histidine to tyrosine substitution at 63rd residue. This amino acid change matched with that reported in Hb M-Saskatoon. This nucleotide change abolished a recognition site for the restriction endonucleaseNlaIII.NalIII digestion of the corresponding -globin DNA amplified from the patient's parents indicated that the mutation was inherited through from his mother. This result shows that the world-wide distribution of Hb M-Saskatoon extends to Indonesia, where it was not previously identified. Possible causes of the unusually severe symptoms observed in the case are discussed.     

TWO CASES OF ACQUIRED TOXOPLASMIC LYMPHADENITIS. Light and Electron Microscopic and Immunohistochemical Studies

We report two cases of acquired toxoplasmic lymphadenitis, one with toxoplasmic cysts and the organisms of Toxoplasma gondii and the other with the organisms only. These cysts and organisms were observed in paraffin-embedded sections, touch smears and ultrathin sections for electron microscopy. Touch smears were especially valuable for the quick and accurate diagnosis of toxoplasmic lymphadenitis. We also studied immature sinus histiocytosis (ISH) in these cases. The predominant cells of ISH were confirmed to be B lymphocytes immunohistochemically, the majority being positive for polyclonal surface IgM. ISH was observed in the perifollicular and paracortical areas surrounding post-capillary venules (PCV), whereas the sinuses were only partially involved.     

Immunohistochemical studies of endocrine cells in heterotopic pancreas

Summary Twenty-one specimens of heterotopic pancreas were investigated using the indirect immunoperoxidase method for insulin, somatostatin, glucagon, pancreatic polypeptide (PP) and gastrin. Ten specimens showed ducts, acini and islets, seven showed ducts and acini, and four showed a ductal component alone. Pyloric gland-like mucous glands were occasionally identified in association with the ductal component. In eight of ten lesions containing islets, the islets were round and had a clearly defined outline with many glucagon cells and either none or a modest number of PP cells (dorsal type). In the remaining two lesions, the islets showed varying sizes and irregular outline with many PP cells and a few or no glucagon cells (ventral type). In either type of islets, insulin and somatostatin were detected, but gastrin cells were absent. Some isolated endocrine cells were also present among the acinar and ductal components. Their occurrence in ducts was more frequent in lesions or areas mainly composed of the ductal compoment than in those with less prominent ductal tissue. In eight lesions a few gastrin cells were found in the ductal component which showed goblet cell metaplasia and pyloric gland metaplasia. An intimate relationship between goblet cell metaplasia and appearance of G cells is noteworthy.     

Morphogenesis of nonpolypoid colorectal adenomas and early carcinomas assessed by cell proliferation and apoptosis

Nonpolypoid neoplasms, as well as ordinary polypoid tumours, are occasionally found in the colorectum. To clarify whether cell kinetic status affects the macroscopic morphology of colorectal neoplasms, we investigated proliferative indices (PI), apoptotic indices (AI), and the expression of apoptosis-related gene products. We examined 110 colorectal neoplasms comprised of 36 polypoid, 38 flat elevated and 36 depressed tumours. According to WHO’s criteria these tumours consisted of 61 adenomas with low grade dysplasia (LGD), 30 adenomas with high grade dysplasia (HGD) and 19 carcinomas with submucosal invasion. Apoptotic cells were detected by TUNEL staining. Proliferating cells and apoptosis-related gene products were assessed by immunohistochemistry for Ki-67, p53, Bcl-2, and Bax antigens. AI were closely associated with macroscopic morphology in adenomas but not in carcinomas. PI were relatively constant among the three macroscopic types in adenomas and carcinomas. Median AI values of polypoid, flat elevated and depressed tumours were 1.8%, 2.1% and 4.6% for adenomas with LGD, 0.8%, 2.4% and 6.2% for adenomas with HGD and 2.9%, 4.0% and 3.6% for carcinomas, respectively. Overall PI were significantly higher in carcinomas than in adenomas with LGD, whereas AI were not different. Although the incidence of expression was significantly higher in carcinomas for p53 and in adenomas for Bcl-2 than the others, the expression of apoptosis-related gene products (p53, Bcl-2 and Bax) was similar among polypoid, flat elevated and depressed tumours. Macroscopic morphology of colorectal adenomas is determined by the apoptosis not by proliferation, and high apoptosis found in depressed adenomas implies their low net growth. Received: 1 July 1999 / Accepted: 17 January 2000     

AN ELECTRON MICROSCOPIC EXAMINATION OF AGE-RELATED CHANGES IN THE RAT LIVER. The Influence of Diet

The influence of age and diet on the ultrastructure of hepatocytes is reported. The following dietary manipulations were investigated: Group 1, fed ad libitum a diet containing 21% protein; Group 2, fed a similar diet but restricted to 60% of the intake of Group 1 from 6 weeks of age onwards; Group 3, restricted from 6 weeks to 6 months of age and thereafter fed ad libitum; Group 4, restriction started at 6 months of age; Group 5, fed ad libitum a diet containing 12.6% protein. In all groups the size of hepatocytes was found not to increase during adult life. The size of hepatocytes in Groups 2 and 4 was the same as or larger than that of the other groups; thus food restriction resulted in a decreased number of hepatocytes. Changes in the structure of some organelles and the accumulation of lipofuscin granules occurred with advancing age and the extent of these age-related changes was less in Groups 2 and 4 than in the other groups. These morphologic findings in conjunction with our previously reported metabolic findings provide a new view of the action of food restriction on the aging process. ACTA PATHOL JPN 38: 1119∼1130, 1988.     

Epstein-Barr virus-positive Hodgkin/Reed-Sternberg-like B cell in non-hodgkin lymphoma: nucleotide sequence of the amplified immunoglobulin heavy-chain variable region gene by the single-cell polymerase chain reaction technique.

We examined nucleotide sequences of Epstein-Barr virus (EBV)-positive Hodgkin/Reed-Sternberg (HRS)-like B cells in a case of diffuse large B-cell lymphoma (DLBCL) and a case of adult T-cell lymphoma (ATL) for single-cell polymerase chain reaction of the immunoglobulin heavy-chain gene variable region (VH gene). HRS-like B cells were scattered in the area irrelevant to the lymphoma infiltrates of DLBCL and in the lymphoma area of ATL. HRS-like B cells were positive for CD20 and CD30 but negative for CD15. EBV presented in HRS-like B cells in both cases but not in any lymphoma cells. VH genes of five HRS-like B cells analyzed in DLBCL were polyclonal and showed in-frame sequences with 0% to 2.8% somatic mutation frequency. In an ATL, VH genes of five HRS-like B cells analyzed were polyclonal and somatically mutated. Four cells carried in-frame rearrangements with 3.5% to 17.7% mutation frequency. One of the VH genes has a one-codon deletion. From the fifth cell, an out-of-frame rearrangement with an insertion and a deletion was obtained. Thus, we showed polyclonal EBV-positive HRS-like B cells in both DLBCL and ATL and that whereas EBV-positive, HRS-like B cells in DLBCL exhibited unmutated and mutated VH gene, those in ATL were found to have a somatically mutated VH gene with/without deletions and/or insertions. The HRS-like B cells may appear because of active EBV infection in a patient who is immunosuppressed from the primary lymphoma.     

CLINICOPATHOLOGICAL STUDY ON SMALL RENAL CELL CARCINOMAS WITH METASTASES

Seven metastasizing small renal cell carcinomas smaller than 30 mm in the greatest diameter were clinicopathologically studied for a better understanding of their characteristic features as compared to those of small tumors without metastases. Grayish-white infiltrating tumors in gross appearance and alveolar or solid microscopic structure consisting of granular or spindle cells and of atypical nuclei were suggestive of having metastases. Two tumors which had positive reactions to the lower nephron markers such as SB A, PNA, and/or DBA were considered to be of lower nephron origin and displayed poor prognosis. ACTA PATHOL. JPN. 37: 947–954, 1987.