Mutagenesis and functional reconstitution of chlamydial major outer membrane proteins: VS4 domains are not required for pore formation but modify channel function.

Chlamidial organisms are obligate intracellular pathogens containing highly antigenic porin-like major outer membrane proteins (MOMPs). MOMP epitopes are of substantial medical interest, and they cluster within four relatively short variable (VS) domains. If MOMPs adopt a beta-barrel fold, like bacterial porins, the VS domains may form extramembranous loops and the conserved regions of the protein may correspond to predicted membrane-located beta-strands. However, molecular studies on native MOMPs have been hampered by the need to culture chlamydiae in eukaryotic host cells and purification and reconstitution remain problematic. In addition, the organisms are difficult to manipulate genetically, and it has also been difficult to functionally reconstitute recombinant MOMPs. To help overcome these problems and improve our understanding of MOMP structure and function, we cloned and expressed C. trachomatis and C. psittaci MOMPs and functionally reconstituted them at the single-channel level. We measured significant functional differences between the two proteins, and by removing and exchanging VS4, we tested the hypothesis that the largest variable domain forms an extramembranous loop that contributes to these differences. Proteins in which VS4 was deleted continued to form functional ion channels, consistent with the idea that the domain forms an extramembranous protein loop and incompatible with models in which it contributes to predicted membrane-located beta-strands. Additionally, the properties of the chimeric proteins strongly suggested that the VS4 domain interacts closely with other regions of the protein to form the channel entrance or vestibule. Our approach can be used to probe structure-function relationships in chlamydial MOMPs and may have implications for the generation of effective antichlamydial vaccines.

Mosaic structure of the human immunodeficiency virus type 1 genome infecting lymphoid cells and the brain: evidence for frequent in vivo recombination events in the evolution of regional populations.

In addition to immunodeficiency, human immunodeficiency virus type 1 (HIV-1) can cause cognitive impairment and dementia through direct infection of the brain. To investigate the adaptive process and timing of HIV-1 entry into the central nervous system, we carried out an extensive genetic characterization of variants amplified from different regions of the brain and determined their relatedness to those in lymphoid tissue. HIV-1 genomes infecting different regions of the brain of one study subject with HIV encephalitis (HIVE) had a mosaic structure, being assembled from different combinations of evolutionarily distinct lineages in p17(gag), pol, individual hypervariable regions of gp120 (V1/V2, V3, V4, and V5), and gp41/nef. Similar discordant phylogenetic relationships were observed between p17(gag) and V3 sequences of brain and lymphoid tissue from three other individuals with HIVE. The observation that different parts of the genome of HIV infecting a particular tissue can have different evolutionary histories necessarily limits the conclusions that can be drawn from previous studies of the compartmentalization of distinct HIV populations in different tissues, as these have been generally restricted to sequence comparisons of single subgenomic regions. The complexity of viral populations in the brain produced by recombination could provide a powerful adaptive mechanism for the spread of virus with new phenotypes, such as antiviral resistance or escape from cytotoxic T-cell recognition into existing tissue-adapted virus populations.

Investigation of population diversity of human immunodeficiency virus type 1 in vivo by nucleotide sequencing and length polymorphism analysis of the V1/V2 hypervariable region of env.

In this study we have analysed variability in the V1 and V2 regions of human immunodeficiency virus type 1 (HIV-1) proviral sequences amplified from lymphoid tissue, brain and other non-lymphoid tissue collected at autopsy from three HIV-1-infected individuals with giant cell encephalitis. We found no evidence for any tissue-specific grouping of variants in the V1/V2 regions, in contrast to previous comparisons of sequences in the V3 region, but consistent with the existence of evolutionarily distinct lineages previously observed in these study subjects by sequence comparisons of the p17gag gene. Examination of inferred amino acid sequences from V1 and V2 revealed no correlations between tissue origin with overall charge, length or number of glycosylation sites. Length polymorphism analysis is a rapid method to compare whole populations of HIV-1 variants within a sample, and provides information on the length and diversity of the V1 and V2 hypervariable regions. Based upon a comparison of 42 individuals with CD4 counts ranging from 802 to < 1 at time of death, we found no evidence for changes in the length of V2 with development of AIDS. Using the number of length variants in the V1 and V2 hypervariable region as a marker of the overall degree of variability within HIV populations, we found no evidence for an increase or a decrease in diversity between those with and without AIDS defining illness.

Investigation of the dynamics of the spread of human immunodeficiency virus to brain and other tissues by evolutionary analysis of sequences from the p17gag and env genes.

The time of spread of human immunodeficiency virus type 1 (HIV-1) from lymphoid to nonlymphoid tissues in the course of infection was investigated by sequence comparisons of variants infecting a range of lymphoid and nonlymphoid tissues from three individuals with AIDS in the pl7gag gene and regions flanking the V1/V2 hypervariable regions. Phylogenetic analysis in both regions revealed several lineages in each individual that contained sequences from both lymphoid and nonlymphoid tissues such as the brain. This observation contrasts strongly with the previously described organ-specific sequences in the V3 region in this study population and other investigations. Although individual pairwise comparisons of relatively short sequences such as p17gag are subject to considerable stochastic error, we found that the diversity of gag sequences in variants from lymphoid tissue was consistently lower than that found among variants amplified from the brain. By estimating mean synonymous pairwise distances in the p17gag region, we were able to make an approximate calculation of the ages of populations in different tissues. Those from lymphoid tissue ranged from 2.65 to 5.6 years in the three study subjects, compared with 4.1 to 6.2 years for variants in the brain. Indeed, variants infecting the brain were no more closely related to each other than they were to variants infecting other tissues in the body. In two of the three individuals, these times of divergence indicate that infection of the brain may have occurred as an early event in the progression to disease, preceding the onset of AIDS by several years. This study is the first in which it was possible to estimate times of diversification in different tissues in vivo and is of importance in understanding the dynamics of the spread of HIV-1 into nonlymphoid tissues and its possible adaptation for replication in different cell types.

In vivo distribution and cytopathology of variants of human immunodeficiency virus type 1 showing restricted sequence variability in the V3 loop.

The distribution, cell tropism, and cytopathology in vivo of human immunodeficiency virus (HIV) was investigated in postmortem tissue samples from a series of HIV-infected individuals who died either of complications associated with AIDS or for unrelated reasons while they were asymptomatic. Proviral sequences were detected at a high copy number in lymphoid tissue of both presymptomatic patients and patients with AIDS, whereas significant infection of nonlymphoid tissue such as that from brains, spinal cords, and lungs were confined to those with AIDS. V3 loop sequences from both groups showed highly restricted sequence variability and a low overall positive charge of the encoded amino acid sequence compared with those of standard laboratory isolates of HIV type 1 (HIV-1). The low charge and the restriction in sequence variability were comparable to those observed with isolates showing a non-syncytium-inducing (NSI) and macrophage-tropic phenotype in vitro. All patients were either exclusively infected (six of seven cases) or predominantly infected (one case) with variants with a predicted NSI/macrophage-tropic phenotype, irrespective of the degree of disease progression. p24 antigen was detected by immunocytochemical staining of paraffin-fixed sections in the germinal centers within lymphoid tissue, although little or no antigen was found in areas of lymph node or spleen containing T lymphocytes from either presymptomatic patients or patients with AIDS. The predominant p24 antigen-expressing cells in the lungs and brains of the patients with AIDS were macrophages and microglia (in brains), frequently forming multinucleated giant cells (syncytia) even though the V3 loop sequences of these variants resembled those of NSI isolates in vitro. These studies indicate that lack of syncytium-forming ability in established T-cell lines does not necessarily predict syncytium-forming ability in primary target cells in vivo. Furthermore, variants of HIV with V3 sequences characteristic of NSI/macrophage-tropic isolates form the predominant population in a range of lymphoid and nonlymphoid tissues in vivo, even in patients with AIDS.

Cancer risk in relatives of patients with common colorectal cancer.

OBJECTIVE: To quantify any risk for colorectal cancer in first-degree relatives of patients with common colorectal cancer and to define possible markers for increased risk. DESIGN: Case-control family study. PATIENTS: Relatives of colorectal cancer patients and of matched control patients from a one-surgeon practice. METHODS: Family medical histories were obtained for 7493 first-degree relatives and 1015 spouses of 523 case-control pairs. Reported diagnoses of colorectal cancer in relatives were verified in 79% of instances. RESULTS: By case-control analysis, the odds ratio was 1.8 (95% CI, 1.2 to 2.7) for one and 5.7 (CI, 1.7 to 19.3) for two affected relatives. By matched analysis of risk in relatives, the increased risk to parents and siblings was 2.1 times greater for case patients than for control patients (CI, 1.4 to 3.1); 3.7 times greater (CI, 1.5 to 9.1) with case patients diagnosed before 45; and 1.8 times greater (CI, 1.2 to 2.9) with case patients diagnosed at 45 years or older; and was independent of gender, type of relative, site of cancer, and type of cancer (single or multiple). The cumulative incidence among first-degree relatives was greater for case patients than for control patients (P < 0.001), and in case patients, greater for those diagnosed before 55 years of age (P < 0.001). The cumulative incidence (+/- S.E.) to age 80 was 11.1% +/- 1.3%, 7.3% +/- 0.8%, and 4.4% +/- 1.0% among relatives of case patients diagnosed before age 45 years, between 45 and 54 years, and at 55 years or older, respectively, and was 2.4% +/- 0.6% in relatives of control patients. CONCLUSIONS: First-degree relatives of patients with common colorectal cancer have an increased risk for colorectal cancer. This risk is greater if diagnosis was at an early and is greater when other first-degree relatives are affected. This increased risk should be considered when formulating screening strategies.

Obstructing carcinomas of the colon.

A series of 908 cases of colonic carcinoma has been analysed to elucidate reasons for the poor prognosis in obstructing colonic cancer. Complete obstruction was present in 148 cases (16.3 per cent), 280 cases (30.8 per cent) had partial obstruction and 480 (52.8 per cent) presented without obstruction. There were fewer Dukes' A tumours in those with complete obstruction (P less than 0.005) and greater numbers of advanced tumours (P less than 0.0005) compared with those without obstruction. This is reflected in a lower curative resection rate of 50.7 per cent in those with obstruction compared with 70.6 per cent in those without obstruction (P less than 0.001). However, after curative resection there was no significant difference in the distribution of tumour stage. Patients with complete obstruction showed a higher incidence of recurrence (P less than 0.01) after curative resection, consequent to an increased incidence of local recurrence (P less than 0.02). Five-year cancer-specific survival for the total series was decreased from 59.1 per cent in patients without obstruction to 31.8 per cent in those with complete obstruction (P less than 0.001). After curative resection there was also a significant reduction in survival (P less than 0.001). It is concluded that completely obstructing colonic cancers are more aggressive than other colonic cancers.

Aggressive angiomyxoma of the female pelvis.

A 42 year old female presented with a 6 year history of a perineal mass. Clinically it had all the features of a perineal hernia. Two previous attempts to repair the 'hernia' had been performed. At operation an aggressive angiomyxoma of the female pelvis was identified and resected via an abdomino-perineal combined approach. The case is discussed with reference to the literature.

Lung recurrence after curative surgery for colorectal cancer.

A total of 1578 patients were treated with potentially curative surgical resection for colon and rectal cancer by one surgeon from 1950 to 1982. Follow-up revealed that 117 (11.5 percent) of 1013 patients with rectal carcinoma eventually presented with clinical evidence of pulmonary recurrence, with or without evidence of spread elsewhere; the corresponding figures for the colon were 20 (3.5 percent) of 565 (P less than 0.001). An analysis of the times to recurrence revealed that half of the lung recurrences were clinically obvious within 32 months for rectal tumors and 34 months for colonic, compared to 22 and 21 months, respectively, for liver recurrences, excluding those with other distant metastases. The slower recurrence rate and the longer survival in patients with recurrences in the lung compared to the liver were statistically significant only for rectal primaries (P less than 0.02 and P = 0.001, respectively). Sixteen patients underwent surgery with curative intention for lung recurrences; four of these remain alive at two, six, 11, and 15 years, and one patient was free of recurrence when he died from other causes 15 months after surgery. The conditional probability survival rate for the 16 patients was 38 +/- 13 percent at five years after recurrence operation.

Changing survival prospects in rectal carcinoma. A series of 1,306 patients managed by one surgeon.

Cancer-specific survival prospects for rectal carcinoma in a series of 1306 patients managed from 1950 to 1979 by one surgeon worsened from 1970 to 1979. The prognosis was worse for all patients treated operatively from 1970 to 1979 compared with 1960 to 1969 (P less than 0.03). After potentially curative resection, survival was worse from 1970 to 1979 compared with 1950 to 1959 (P less than 0.02) and 1960 to 1969 (P less than 0.01), respectively; the corresponding five-year survivals were 72.5 percent, 72.3 percent, and 61.5 percent. The curative resection rate for the three decades was similar (66 to 70 percent). An increase in the incidence of Dukes' Stage C tumors from 23.3 percent to 32.3 percent (P less than 0.01) explains, at least partly, the decreased survival. The worsened survival prospects were not accounted for by changes in referral pattern, tumor site, or in the proportion of sphincter-saving resections performed. The worsening was paradoxically paralleled by earlier symptomatic presentation (P less than 0.001). Analyses of other Australian data are required to test the hypothesis that the worsened survival prospects are consequent to altered tumor biologic aggressiveness, possibly related to differences in the causal factors operating over the 30-year study period.

The outcome of patients with ulcerative colitis managed by subtotal colectomy.

A total of 1,494 patients with a diagnosis of ulcerative colitis were managed by one surgeon between 1950 and 1981. Subtotal colectomy was performed upon 286 patients of whom 82 had a primary and 65 a secondary ileorectal anastomosis. There was no significant difference in the postoperative mortalities between patients treated by subtotal colectomy (8.7 per cent) and a group of 71 patients treated by proctocolectomy (9.9 per cent). The postoperative mortality after primary and secondary ileorectal anastomosis was 6.9 and 1.5 per cent, respectively. Rectal excision because of disease activity was required in 109 patients after subtotal colectomy (13 after primary and nine after secondary ileorectal anastomosis) with a postoperative mortality of 0.5 per cent. The probability of rectal excision was 21 per cent at 12.6 years after primary anastomosis and 19 per cent at 16.2 years after secondary ileorectal anastomosis. Carcinoma of the rectum has developed in 11 (3.8 per cent) of the patients after subtotal colectomy. The probability of having carcinoma of the rectum develop was 17.1 per cent at 27 years after primary anastomosis and 20.0 per cent at 24 years after secondary ileorectal anastomoiss, the time beyond which no disease has yet been diagnosed in either group. The disease was advanced in stage and of high histologic grade with a median cancer specific survival time of 14 months.

Prognostic significance of the absolute lymphocyte count in acute pancreatitis.

The absolute lymphocyte count was calculated with 48 hours of admission in 154 patients with acute pancreatitis. Twenty-three patients had severe pancreatitis and 131 patients had mild pancreatitis. The mean absolute lymphocyte count for the severe group was 880 +/- 102 cells/mm3 and for the mild group, 1,115 +/- 116 cells/mm3. The absolute lymphocyte count accurately predicted 78 percent of the severe attacks and 86 percent of the mild attacks.

Total and subtotal colectomy for colonic obstruction.

Colectomy (total or subtotal) is not the operation of choice for elective colonic cancer unless the patient is under 50 years of age, is undergoing curative resection, and has associated adenomatous polyps. Routine colectomy is not supported when a partial obstruction prevents preparation of the colon and interferes with proximal colonic examination. Colectomy (especially subtotal) is acceptable for the acutely obstructed colon but it is technically demanding and requires experience in patient selection.

Post-proctectomy perineal hernia: case report and discussion.

Perineal hernia is a rare complication of pelvic surgery. Women are more commonly affected, and development of the condition is usually gradual. Although the true incidence of this problem is unknown, few cases are severe enough to require operative repair. We report the experience of one of the authors (E.S.R.H.) in post-proctectomy perineal hernia. One case required surgical repair and is presented, with a discussion of the operative management.

Local recurrence after potentially curative resection for rectal cancer in a series of 1008 patients.

Local recurrence and its related mortality after potentially curative resection for rectal cancer have been analysed in a series of 1008 patients managed by one of the authors. Nine hundred and thirty-four were available for analysis of recurrence. One hundred and seven (11 per cent) patients developed local recurrence without evidence of systemic spread and 84 (9 per cent) both local and systemic recurrence. Local recurrence was less common (14 per cent) after resection of tumours of the upper third of the rectum compared with the middle (21 per cent) (P = 0.02) or lower thirds (26 per cent) (P less than 0.001). Local recurrence was related to both tumour stage and differentiation (P less than 0.001). There was no significant relationship between local recurrence and tumour size or the type of curative resection performed, restorative or non-restorative. The distal margin of clearance after restorative resection did not influence the local recurrence rate. Of patients who developed metastases recurrence was evident within 2 years in 60 per cent. Three hundred and thirty-two patients died from recurrence, 91 (27 per cent) with evidence of local recurrence only, 80 (24 per cent) with combined local and systemic recurrence and 161 (48 per cent) with evidence of systemic spread only. The corresponding median survivals were 35, 34 and 39 months.

T-antigen expression by peanut agglutinin staining relates to mucosal dysplasia in ulcerative colitis.

Staining of 326 rectal mucosal biopsies from ulcerative colitis patients with peanut agglutinin (PNA), which binds to the T-blood group antigen and has been claimed to reflect a cancer-associated mucin alteration, showed highly significant direct associations with mucosal dysplasia (P less than 0.001), disease activity (P less than 0.001), and subsequent development of rectal cancer in a smaller series of patients (P = 0.005). Staining for normal colonic mucin by the Dolichos biflorus (DBA) lectin related significantly and inversely to dysplasia. Intense normal colon mucin staining by DBA related significantly (P less than 0.025) to long disease duration and to subsequent development of cancer (P = 0.02). The latter association is based on a small number of patients only and is not considered conclusive evidence, but may provide a link with goblet-cell hyperplasia. The authors conclude that although T-antigen expression relates to dysplasia, the findings of "false" positive and negative rates of 22 and 33 percent respectively, make it unlikely that staining of biopsy sections for the T-antigen by peanut agglutinin will contribute materially to routine assessment for dysplasia and cancer risk prediction in patients with ulcerative colitis.