ACE and ACE2: their role to balance the expression of angiotensin II and angiotensin-(1-7).

The discovery of angiotensin-converting enzyme 2 (ACE-2) has revealed a far more complex enzymatic cascade that may influence the renin-angiotensin system within the kidney, specifically the expression of the functional products angiotensin II (Ang II) and Ang-(1-7). The regulation of this critical system involved in blood pressure control must now encompass the integral relationship of ACE and ACE-2 activities.

Regulation of cell survival during B lymphopoiesis: increased pre-B cell apoptosis in CD24-transgenic mouse bone marrow.

CD24 (heat-stable antigen) is expressed in a developmentally regulated fashion by B cell precursors in mouse bone marrow (BM), but its role in B lymphopoiesis remains obscure. A slight overexpression of CD24 in transgenic (Tg) mice leads to depletion of B lymphoid cells in BM. The present study examines whether CD24 is involved in apoptotic selection of B lineage cells under normal microenvironmental conditions in vivo. Double immunofluorescence labeling and flow cytometry have been used to quantitate the apoptotic rates of phenotypically defined B cell populations in BM of CD24-Tg mice. Apoptosis of pre-B cells expressing cytoplasmic mu heavy chains of IgM but lacking surface (s)IgM was increased both ex vivo and in short-term culture, while the number of pre-B cells was halved compared to BM of normal mice. In contrast, B220+mu- pro-B cells and sIgM+ B lymphocytes showed no significant change in either apoptosis or number. The findings provide evidence that CD24 can play a role in vivo in modulating pre-B cell apoptosis, a quality control checkpoint in B cell development.

Cross-linking the murine heat-stable antigen induces apoptosis in B cell precursors and suppresses the anti-CD40-induced proliferation of mature resting B lymphocytes.

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes, the co-stimulation of CD4+ T cells, and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells, but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together, these data implicate HSA as a potent negative regulator of B cell development and activation.

Reduction of early B lymphocyte precursors in transgenic mice overexpressing the murine heat-stable antigen.

To study the role of the murine heat-stable Ag (HSA) in lymphocyte maturation, we generated transgenic mice in which the HSA cDNA was under the transcriptional control of the TCR V beta promoter and Ig mu enhancer. The HSA transgene was expressed during all stages of B lymphocyte maturation. Expression was first detected in the earliest lymphoid-committed progenitors, which normally do not express HSA, and subsequently reached the highest levels in pro- and pre-B cells. In bone marrow, the number of IL-7-responsive clonogenic progenitors was < 4% of normal, whereas the frequency of earlier B lymphocyte-restricted precursors, detectable as Whitlock-Witte culture-initiating cells, was normal. Pro- and pre-B cells detected by flow cytometry were reduced by approximately 50% relative to controls. Mature splenic B cells were also reduced but to a lesser extent than in marrow, and their response to LPS stimulation was impaired. Reconstitution of SCID and BALB/c-nu/nu mice with HSA transgenic marrow indicated that the perturbations in B lymphopoiesis were not caused by a defective marrow microenvironment or by abnormal T cells. Our previous studies showed elevated HSA expression throughout thymocyte development, which resulted in a profound depletion of CD4+CD8+ double-positive and single-positive thymocytes. Together, these results indicate that HSA levels can determine the capacity of early T and B lymphoid progenitors to proliferate and survive. Therefore, HSA could serve as an important regulator during the early stages of B and T lymphopoiesis.

Clinical and pathological evaluation of large loop diathermy excision of the transformation zone.

In order to make accurate diagnosis and to carry out treatment of cervical preneoplastic disease, large loop diathermy excision of the transformation zone was performed in 98 patients. The colposcopic assessment was indicated by abnormal smear or history of treatment for preneoplastic changes. The entire transformation zone could be excised in one piece in 90% of cases. Histological examination of the specimens confirmed dysplasia in 89% of patients and in 4 cases invasive cervical disease was revealed. The ectocervical and endocervical excision margins were free of dysplastic epithelium in 68% of cases. Compared to traditional cone biopsy, the new method is cheaper and more simple. Loop diathermy excision of the transformation zone can be performed in local anaesthesia as an out-patient procedure and there is no need for postoperative hospitalization. By reducing the number of general anesthesia, the workload in gynaecological theatres and by eliminating the need for postoperative hospital stay the method substantially contributes to the improvement of the hospital budget.

Identification of a secondary Fc gamma RI binding site within a genetically engineered human IgG antibody.

Although human IgG2 is not cytophilic, we have shown previously that an IgG2 antibody expressing the sequence PLLGG (underline = substitution) spanning CH2 domain residues 233-237 (Eu numbering) displayed IgG1-like Fc gamma RI binding activity. In contrast, IgG1 PLLGG exhibited 3-fold less affinity, whereas IgG2 ELLGG was 3-fold more active than native IgG1. These results suggested that additional site(s) conferred enhanced binding properties to the engineered, cytophilic IgG2 variant. These sites were shown to reside in the IgG2 CH2 domain, since the IgG1 CH2 module did not have enhanced activity in a panel of hybrid IgG1/IgG2 antibodies. To map these sites further, human IgG1 and IgG2 constant region gene segments were modified to allow reciprocal COOH-terminal half segment exchanges of CH2 exons. These were cloned into a pSV2neo expression vector bearing a rearranged MOPC 315 heavy chain variable region gene and transfected into a MOPC 315 heavy chain deletion mutant. The dinitrophenol affinity-purified IgGs were radiolabeled and assessed for Fc gamma RI binding activity in direct binding assays using U937 cells. The COOH terminus of the IgG2 CH2 domain was found to contain accessory site(s) since it enhanced the binding properties of both IgG1 PLLGG and native IgG1. In contrast, grafting of the COOH terminus of the IgG1 CH2 domain onto IgG2 PLLGG and IgG2 ELLGG diminished their cytophilic activity. The amino acid responsible for the enhancing properties of the COOH terminus of the IgG2 CH2 domain was shown to be threonine 339, since IgG1 PLLGG/Thr339 displayed increased Fc gamma RI binding affinity. Kinetics studies revealed that this is accomplished through an increase in the forward rate constant of the IgG-Fc gamma RI interaction.

Identification of the Fc gamma receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies.

To characterize the region on human IgG1 responsible for its high-affinity interaction with the human Fc gamma receptor class I (Fc gamma RI), we have analyzed the binding properties of a series of genetically engineered chimeric antidinitrophenyl antibodies with identical murine antibody combining sites and hybrid IgG1/IgG2 human constant (C) regions. In addition, we have investigated a panel of reciprocally point-mutated IgG1 and IgG2 chimeric antibodies to identify the amino acid residues that confer cytophilic properties to human IgG1. Our data unambiguously indicate that cytophilic activity of IgG1 is an intrinsic property of its heavy-chain C region 2 (CH2) domain. We report that the entire sequence spanning residues 234-237 (LLGG) is required to restore full binding activity to IgG2 and IgG4 and that individual amino acid substitutions failed to render IgG2 active. Nevertheless, the reciprocal single point mutations in IgG1 either significantly lowered its activity or abolished it completely. Finally, we observed that an IgG2 antibody containing the entire ELLGGP sequence (residues 233-238) was more active than wild-type IgG1. This finding suggests that in addition to the primary contact site identified in the N terminus of the gamma 1 CH2 domain, secondary sites involving residues from the C-terminal half of the domain may also contribute to the IgG1-Fc gamma RI interaction.

Cyclosporin in the treatment of steroid-responsive and steroid-resistant nephrotic syndrome in adults.

The effect of cyclosporin on proteinuria was studied in 11 patients with steroid-responsive nephrotic syndrome (10 minimal change nephropathy, one IgM nephropathy) and in four patients with steroid-resistant nephrotic syndrome from focal segmental glomerulosclerosis. Cyclosporin (mean initial dose 7.7 mg/kg per day) produced a complete remission of proteinuria in 15 nephrotic episodes in the ten patients with minimal-change nephropathy after a mean 14.3 days (range 7-23 days) of therapy. All patients remained in remission while receiving cyclosporin (mean duration of follow-up 147 days; range 40-230 days). However, when cyclosporin was discontinued on nine occasions in five patients, all relapsed after a mean 47.8 days (range 7-180 days). Four of the five patients were subsequently rechallenged with cyclosporin and all responded. Maintenance cyclosporin therapy to prevent relapse was not associated with any adverse effects, and there was no significant difference between the creatinine clearance before and after 30 days of therapy (86.9 +/- 19.3 and 81.7 +/- 23.5 ml/min respectively, P greater than 0.1). The patient with steroid-responsive IgM nephrotic syndrome did not respond to cyclosporin, and there was no significant effect of cyclosporin on proteinuria in the four patients with FSGS. Cyclosporin is an effective agent for the treatment of patients with frequently relapsing minimal-change nephropathy who became steroid dependent when cyclophosphamide is contraindicated. However, unlike cyclophosphamide, long-term remissions which persist after treatment is withdrawn are not obtained, and patients may be said to be cyclosporin dependent.

Postperfusion xenon-133 ventilation scintigraphy with a narrow window.

Xenon-133 postperfusion lung scintigraphy using 10% windows was compared with standard posterior preperfusion 133Xe ventilation scanning in 33 patients. The postperfusion 133Xe study identified all major defects and washout abnormalities. In five patients, the assessment of match or mismatch of defects was improved because of optimal positioning of the postperfusion ventilation study. Computer subtraction of background technetium-99m macroaggregated albumin activity improved detection of mild washout abnormalities in eight patients but did not change the diagnostic category in any case. Postperfusion ventilation scanning using 10% windows (with or without background computer subtraction) is an alternative to preperfusion ventilation scanning for major V/Q abnormalities.