Early intervention and disease modification in atopic dermatitis-the current state of the field and barriers to progress.

Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease representing a major source of global disability burden. Disease-modifying therapies are showing promise in chronic inflammatory disorders such as rheumatoid arthritis and Crohn's disease with method and timing of initial treatment impacting long-term disease outcomes. Whether disease-modifying therapies, specifically those used as an early interventional approach, impacts disease course and comorbidity development in AD is not well-understood. We reviewed the progress in disease modification strategies, emphasizing early intervention approaches in common (or proto-typical) inflammatory diseases. Although more common in other fields, disease modification approaches are becoming increasingly investigated in dermatology, though studies in AD are lacking. Despite significant limitations in ongoing and completed studies, early data are promising and suggest that both the choice and timing of early intervention approach can affect long-term disease course and comorbidity development. To best improve AD patient outcomes, more research is needed to further explore the impact of early disease-modifying therapies. Future studies should focus on identifying the most effective approaches and extend the early results to a more inclusive set of comorbidities and longer-term outcomes.

Increased mutation frequency following treatment with cancer chemotherapy.

The relationship between somatic mutation and cancer was studied by measuring in vivo mutation frequency and in vitro mutability using lymphocytes from 28 untreated adult patients with solid tumors, 14 untreated patients with lymphoma, and 27 patients with solid tumors or lymphoma who had been treated with chemotherapy and/or radiotherapy. In vivo mutation frequency in untreated patients did not differ from that of controls, except perhaps in patients with lymphoma, who showed a slight increase. Lymphocytes from untreated patients with solid tumors or lymphoma did not show a greater increase in mutations induced after X-radiation or UV radiation than did lymphocytes from controls. For all the untreated patients, the geometric mean mutation frequency was 6.72 X 10(-6), and it was significantly increased to 19.57 X 10(-6) following chemotherapy and 34.40 X 10(-6) following chemotherapy and radiotherapy. The results suggest that excessive systemic exposure to mutagens or inherent susceptibility to mutagenesis are not important etiological factors in at least the majority of patients with cancer. The mutations produced by treatment may be related to the late side effects of therapy such as second neoplasms.

The cells of origin of lymphocyte clones grown by limiting dilution.

A high proportion of the mononuclear cells separated from peripheral blood by Ficoll-Hypaque centrifugation show extensive proliferation when cloned at limiting dilution. The nature of clone-forming cells ( CFCs ) and the cells generated during clone formation were studied by cytotoxicity with antibody and complement and by immunofluorescence. Cytotoxicity prior to cloning with OKT3 plus OKT11 eliminated 99.34% of clones, indicating that virtually all CFCs are T-lymphocytes, and cytotoxicity with OKT4 or OKT8 indicated that helper and suppressor subclasses each contribute approximately half of the CFCs . Immunofluorescence of cells proliferating in clones confirmed that all clones were T-lymphocytes and showed that 47% were OKT4 positive and 47% were OKT8 positive; 6% were negative with both OKT4 and OKT8. The results indicate that in the nearly optimal conditions for proliferation provided by limiting dilution, clones arise entirely from T-lymphocytes and clone formation is a property of a variety of lymphocyte subclasses.

An improved method for the growth of human lymphocyte colonies.

An improved method has been developed for growth of human lymphocyte colonies in agar. The method is a single-step assay involving the addition of irradiated lymphocytes which do not themselves form colonies but which stimulate a small number of non-irradiated cells to do so. Over the range 0 to 1 x 10(5) target lymphocytes, there is a linear relationship between number of lymphocytes cultured and number of colonies produced. Technical factors which influence colony growth include osmolality of the medium, foetal calf serum concentration, agar concentration, PHA type and concentration, and 2-mercaptoethanol. Colony formation is not affected by using homologous irradiated cells from an individual of the same age. For normal individuals aged 11 to 45, colonies of 20-200 cells were grown to a plating efficiency of 2.18%, with a range 1.28-4.00%. The advantages of the method are its simplicity, the linear relationship between cells plated and colonies formed, which enables the method to be used as a quantitative assay, and the ability to determine whether an observed abnormality is due to an abnormality of colony forming cells or of irradiated stimulating cells.

A proliferative defect of marrow cells in experimental chronic hypoplastic marrow failure (aplastic anaemia).

Marrow cells from control mice and mice with chronic hypoplastic marrow failure (CHMF, aplastic anaemia) were grown in tissue culture and growth was assessed by measuring the number of cells/colony and uptake of 3H-thymidine/colony. Cells from mice with CHMF showed impaired proliferation in response to colony stimulating factor. Mixing experiments suggested that the impairment of proliferation was not dut to alteration in suppressor or helper cells but to an intrinsic lesion of the marrow cells themselves.

Cloning of human lymphocytes using limiting dilution.

A simple technique for cloning lymphocytes at limiting dilution in microwells has been developed using phytohaemagglutinin, irradiated cells and conditioned medium containing interleukin-2. For freshly isolated peripheral blood lymphocytes, the mean frequency of clone forming cells (CFC) was 29.4% and for continuously cultured lymphocytes it was 25.5%. Quantitative considerations, mixing experiments involving HLA-A2 positive and negative cells and replating experiments indicated that growth in most of the positive microwells originated from a single cell which had a very high self-renewal capacity and underwent approximately 15 divisions during the 14 days of culture. The high plating efficiency and sustained growth observed with this technique suggests that it is the method of choice for enumerating clone forming cells or for isolating clones.

Age-related variations in human lymphocyte DNA.

The molecular weight of DNA from human peripheral lymphocytes has been measured on alkaline sucrose density gradients. The number average molecular weight (MN) of DNA is found to decrease as the age of the donor increases. This result is discussed with reference to lesions in DNA, both repairable and non-repairable, which may accumulate with age.

Radiation sensitivity of human lymphocytes.

A simple and rapid in vitro technique to assess the sensitivity of human lymphocytes to X- and UV-radiation is described. Peripheral blood lymphocytes are cloned in agar and colony survival is used as the end point. Survival down to a fraction of approx. 0.02 can be measured accurately. The D10 for X-radiation is approx. 165 rad and the initial portion of the survival curve is linear with no evidence of a threshold. The D10 for UV-radiation is approx. 50 erg/mm2. The technique should be of value both for general radiobiological studies and for detecting individuals having abnormalities of DNA repair.

Methods for study of mutations and mutagenesis in human lymphocytes.

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.

Swallowing problems in post irradiated NPC patients.

We present three patients with Nasopharyngeal Carcinoma (NPC) developing swallowing problems after radiotherapy as the primary modality of treatment. All patients had advanced stage NPC presenting with enlarged neck nodes and underwent radical external beam radiotherapy. All three patients had both CN X and CN XII palsies and had difficulty in both the oral and pharyngeal phases of swallowing. None of them has any clinical or radiological evidence of local recurrence in the post nasal space and neck or metastasis to the skull base. One patient underwent cricopharyngeal myotomy with epiglottopexy and hyoid suspension which failed and subsequently underwent laryngectomy. Another patient had medialisation thyroplasty and the third underwent a percutaneous endoscopic gastrostomy (PEG).

Heritable fragile sites on human chromosomes. V. A new class of fragile site requiring BrdU for expression.

A new fragile site at 10q25 is described, representing a new class of fragile site that requires bromodeoxyuridine (BrdU) in the culture medium for expression. This new fragile site is present in approximately one in 30 of the Australian population; it has only been observed in heterozygotes.

Chronic hypoplastic marrow failure and residual injury.

An experimental model of chronic marrow failure can be produced in mice by treatment with a short course of busulphan. This results in permanent partial marrow failure which may later progress and become complete. The underlying lesion appears to be stem cell damage which leads to failure of proliferation of stem cells and their progeny. Other drugs can produce the same lesion and alkylation of DNA may be the mechanism.

Lymphocyte DNA in aplastic anaemia.

Using ultracentrifugation on alkaline sucrose density gradients the DNA of lymphocytes from 14 patients with aplastic anaemia and 23 controls was studied before and after exposure to bleomycin, an agent known to cause strand breaks in DNA. Before exposure to bleomycin the DNA from aplastic patients had more strand breaks than the DNA from the controls of similar ages. Following exposure to bleomycin an abnormal number of DNA strand breaks was produced in 10 of 14 patients and this molecular evidence of drug sensitivity correlated well with the sensitivity of the proliferating lymphocytes to bleomycin in tissue culture. Furthermore, two relatives of patients with aplastic anaemia showed similar abnormality of DNA before and after exposure to bleomycin and increased sensitivity to bleomycin in tissue culture. These results suggest that an abnormality of DNA structure and/or repair may be present in some patients with aplastic anaemia.

Reduced lymphocyte colony formation with age.

The lymphocyte colony forming ability of 83 normal healthy individuals aged 17-89 was studied. The total lymphocyte count did not alter with age, but there was a decline in the colony forming ability of cells from people with increasing age (r = -0.68, P less than 0.001). This age related defect was further investigated in 11 mixing experiments using paired young and old individuals. It was found that the defect in aged individuals was due primarily to a defect in the number and/or proliferative ability of the colony forming cell.

Leukemic reticuloendotheliosis. A failure of monocyte production.

In five cases of leukemic reticuloendotheliosis studied, profound monocytopenia was demonstrable. In 48 normal subjects, monocyte counts ranged from 215 to 921 per cubic millimeter, whereas in the five patients with leukemic reticuloendotheliosis, the counts ranged from 0 to 17 per cubic millimeter. The properties of the "hairy" cell are consistent with those of an incompletely developed monocyte. The susceptibility to infection, characteristics of these patients, may result from a lack of normal monocytes in blood and tissues.

Sensitivity of human lymphocytes to bleomycin increases with age.

The sensitivity of human peripheral blood lymphocytes to bleomycin and mitomycin-C was assessed by measuring the inhibition of phytohemagglutinin stimulated proliferation. The sensitivity to bleomycin, and not to mitomycin-C, increased with the age.

Effective use of blood in elective surgical procedures.

The preoperative requests for crossmatching of blood in elective surgical procedures were studied at the Flinders Medical Centre, South Australia. The study revealed that surgeons order crossmatched blood on the basis of habit. This led to considerable time-expiry of blood, and to unnecessary use of laboratory personnel's time and reagents. The statistical information collected during the study was used to educate the surgeons to change their blood-ordering practice. In procedures where excessive blood loss is unlikely to occur, as a stand-by, a "group-and-screen" procedure was substituted for crossmatching. A firm recommendation for maximum blood order in elective surgical procedures was also made. It is estimated that this approach would save approximately $80,000 per year per 350-bed general hospital in Australia.

A LISS spin enzyme method for the detection of red cell antibodies and its use in routine antibody screen procedures.

A technique for performing the enzyme phase of the antibody screen on red cells suspended in low-ionic-strength-salt solution (LISS) is described. The reliability of this LISS spin-enzyme (LSE) technique was compared with the two-stage papain-tile method, in the detection of 62 previously identified enzyme reacting antibodies. All 62 antibodies examined were detected by the LSE method, and no false-positive reactions were found. Using LSE and papain-tile methods in parallel, further assessment was obtained by screening 2000 sequential blood samples under routine service conditions. Fifty-six blood samples contained alloantibodies, of which 43 reacted by both methods, eight by the LSE method only, and five by the papain-tile method only. It was concluded that the LSE method was comparable to the papain-tile method.